Rat epididymal adipose tissue was found to contain at least two enzymes which are able to catalyze the hydrolysis of tributyrin. These activities were differentiated by their heat stability and sensitivity to inhibition by sodium fluoride. About 80% of the tributyrinase activity in epididymal adipose tissue appears to be identical with a Tween hydrolase which has been previously partially purified. This enzyme was further purified by gel filtration on Sephadex G-200, and its electrophoretic mobility was shown to be comparable with that of the major tributyrinase activity in crude extracts of adipose tissue. The substrate specificity of this enzyme indicates that it is probably not a true lipase (EC 3.1.1.3). The remaining tributyrinase activity was not fully investigated, but preliminary experiments indicate that it sediments with the microsomal fraction of adipose tissue extracts.