Isolated frog muscles were exposed to Ringer’s solution of widely varying pH. In the presence of oxygen they remained in good condition for a long time and continued to contract well at hydrogen-ion concentrations many times greater (up to x 200) than that of their normal environment in the body. If the Donnan equilibrium, which is believed to govern the K and Cl ion ratios across the fibre membrane, applied also to H and HCO
3
ions, the internal pH in these circumstances would be 3.8 or less. It is difficult to believe that the contractile mechanism would function so well under such conditions, but the question could be examined experimentally as follows. Muscles in oxygen-free Ringer at pH 7.4 to 3.3 were stimulated in a regular series of twitches to complete exhaustion, and the total tension developed was used to measure the energy liberated. If the energy was less than 0.4 cal/g muscle it could have been derived solely from the splitting of creatine phosphate and other phosphorus compounds; if it was greater than 0.4 cal/g muscle it must have been obtained in part from lactic acid production. The formation of lactic acid in response to stimulation ceases when the internal pH falls below about 6.3; but experiments show that at external pH 6.0 adjusted by phosphate buffers, lactic acid can be produced in practically normal amount, while some lactic acid can be formed even when the external pH is as low as 4.5. When muscles are stimulated in a medium saturated with 100% CO
2
and buffered with bicarbonate, there is seldom evidence of lactic acid formation at any external pH (from 6.8 downwards). The CO
2
itself appears to reduce the internal pH to about the critical level below which lactic acid production is inhibited. At lower CO
2
percentages (50% or less, in nitrogen) some lactic acid can be produced at all external pH’s, from 5.1 upwards. If the hydrogen-ion ratio across the fibre membrane were governed by the Donnan equilibrium, the internal pH of a normally excitable muscle would have to be at least 1.2 less than that of the outside fluid. At external pH 6.0 the internal pH would then be 4.8 or less, yet the nearly normal production of lactic acid shows that it must have been well above 6.3, while at external pH 4.5 the internal pH would not be greater than 3.3, yet some lactic acid was formed, so it cannot in fact have been less than 6.0 (allowing 0.3 for the increased alkalinity due to phosphagen splitting). When phosphate buffers are used, the internal pH certainly falls to some extent when the external pH is lowered, but far less than prescribed by the Donnan equilibrium. With CO
2
-bicarbonate buffers, there is no sign that the internal pH depends on anything but the partial pressure of CO
2
. Other evidence is considered, particularly that obtained by using an intracellular glass electrode (Caldwell, with crab fibres). The conclusion is that in normally excitable muscle the Donnan equilibrium does not control, and does not greatly influence, the distribution of hydrogen ions across the fibre membrane. In resisting diffusion and potential gradients the muscle fibre probably maintains its own internal pH, at least to a large extent, by active metabolic effort, If so, since CO
2
penetrates freely, the internal HCO
3
-ion concentration also must be actively maintained. When the controlling mechanism fails, the contractile function of the muscle deteriorates. The observed variability of muscles exposed to abnormal external conditions may depend on differences in their capacity to maintain their internal state.
Lactic Acid Permeability of Aquaporin-9 Enables Cytoplasmic Lactate Accumulation via an Ion Trap
