The acyl phosphatase activity of glyceraldehyde 3-phosphate dehydrogenase has been examined at 25°. This activity can conveniently be divided into three categories depending upon the steric bulk in the acyl group of the substrate, that of: (1) acetyl phosphate which follows apparent second-order kinetics, Km being too large to measure; (2) propionyl, butyryl, isobutyryl, and isovaleryl phosphate which obey normal Michaelis-Menten kinetics; and (3) the highly branched compounds, trimethylacetyl and 3,3-dimethylbutyryl phosphate, which are not substrates. Arsenate increases the rate of hydrolysis of the compounds in group 2, showing the rate-determining step for those compounds to be deacylation of the acyl enzyme intermediate. Methyl phosphate, however, was found to be a poor catalyst for deacylation of the enzyme. This and other observations support a general base mechanism for deacylation of glyceraldehyde 3- phosphate dehydrogenase. The highly branched compounds bind to the enzyme since they inhibit acetyl phosphatase activity. In addition, compounds in groups 2 and 3 inhibit the dehydrogenase activity. This inhibition was found to be sigmoidal and was related to a cooperativity effect on binding to the enzyme. A plot of log k3 (deacylation) vs. Es, the Taft steric effects constants, had a slope of 1.1. © 1969, American Chemical Society. All rights reserved.