Effects of wheat germ lipase on S-acyl and disulfide derivatives of thiamine were investigated. When the derivatives were incubated with lipase, thiamine liberated from diacetyl-thiamine (DAT) and thiamine-8-(methyl-6-acetyldihydrothioctate) disulfide (TATD). No thiamine liberation was observed when the other derivatives and denaturated enzyme were used in the experiment. The quantitative assay of acetyl group of both derivatives indicated that the acetyl contents of the derivatives decreased during the incubation with enzyme and there was correlations between thiamine liberation and decrease of acetyl contents. From the results, it was suggested that the thiamine liberation was caused by the enzymatic hydrolysis of acetylthioester containing in both DAT and TATD, and especially, the liberation from TATD was caused by the split of its disulfide linkage with SH radical which produced from the hydrolysis of acetylthioester containing in its thioctate moiety.