1. A reaction mixture, made from pig heart pyruvate oxidase, hydroxypyruvate, thiamine pyrophosphate, and magnesium chloride, is described in which thiamine pyrophosphate is almost quantitatively converted into 2-(1,2-dihydroxyethyl)-thiamine pyrophosphate (thiamine pyrophosphate-activated glycolaldehyde). This product can be isolated by ion exchange chromatography.2. 2-(1,2-dihydroxyethyl)-thiamine pyrophosphate can also be isolated from reaction mixtures of yeast transketolase with hydroxypyruvate, thiamine pyrophosphate, and magnesium chloride, but in very low yield.3. From non-enzymatic reaction mixtures containing thiamine pyrophosphate and a great excess of glycolaldehyde, a compound could be isolated, the tested properties of which were found to be the same as those of enzymatically prepared 2-(1,2-dihydroxyethyl)-thiamine pyrophosphate.4. 2-(1,2-dihydroxyethyl)-thiamine pyrophosphate gives rise to only 55% of the thiochrome which is produced from the same quantity of thiamine pyrophosphate. This behaviour can be used for the determination of relative concentrations of thiamine pyrophosphate and 2-(1,2-dihydroxyethyl)-thiamine pyrophosphate in mixtures.