The effects of ionic strength and cationic valency of the fluid medium on the surface potential and dextran-induced aggregation of red blood cells (RBC's) were investigated. The zeta potential was calculated from cell mobility in a microelectrophoresis apparatus; the degree of aggregation of normal and neuraminidase-treated RBC's in dextrans (Dx 40 and Dx 80) was quantified by microscopic observation, measurement of erythrocyte sedimentation rate, and determination of low-shear viscosity. A decrease in ionic strength caused a reduction in aggregation of normal RBC's in dextrans, but had no effect on the aggregation of neuraminidase-treated RBC's. These findings reflect an increase in electrostatic repulsive force between normal RBC's by the reduction in ionic strength due to (a) a decrease in the screening of surface charge by counter-ions and (b) an increase in the thickness of the electric double layer. Divalent cations (Ca++, Mg++, and Ba++) increased aggregation of normal RBC's in dextrans, but had no effect on the aggregation of neuraminidase-treated RBC's. These effects of the divalent cations are attributable to a decrease in surface potential of normal RBC's and a shrinkage of the electric double layer. It is concluded that the surface charge of RBC's plays a significant role in cell-to-cell interactions.