Transketolase has been purified from rat liver. The final product is judged to be homogeneous by the criteria of gel filtration on Sephadex G-200 and Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The estimated Mr's are 139,000 for the native protein and 69,000 for the dissociated subunits. The purified enzyme does not require either Mg2+ or thiamine pyrophosphate for optimum activity. The best C2-donor substrate is D-xylulose 5-phosphate, with Km = 25 microM. D-Fructose 6-phosphate is an active C2-donor, but beta-hydroxypyruvate was found to be inactive, even at high concentration. The product of the C2-transfer from D-xylulose 5-phosphate or D-fructose 6-phosphate to D-ribose 5-phosphate has been identified as D-sedoheptulose 7-phosphate. Neither D-glucose 6-phosphate nor D-arabinose 5-phosphate showed C2-acceptor activity. This enzyme does not appear to be responsible for the formation of octulose 8-phosphates which have been detected in liver extracts.