Cholesteryl ester hydrolase (CEH) was measured at 32 degrees C and 37 degrees C, and with and without cofactors for stimulation of cyclic AMP-dependent protein kinase, in 104,000 X g supernatants from rats aged 14-365 days. Activity at the two temperatures was also partially resolved by cation exchange FPLC. Total specific activity of CEH was relatively constant, with or without addition of cofactors, from 14 to 47 days, during which time temperature labile CEH was a very small fraction of total CEH activity. At later times, 51-150 days, activity was increased as much as two-fold, both with and without cofactors, with most of the increase occurring in the temperature labile fraction. Activation of temperature stable and temperature labile activities, where present, by protein kinase cofactors could be demonstrated in all age groups, but was highly variable as a function of age and protein concentration used in the assay. Apparent induction of temperature labile activity over the interval 47-51 days coincides with reported increases in testosterone synthesis and first appearance of spermatozoa in the testis. This and other lines of evidence suggest unique roles for these enzymes in regulation of availability of free cholesterol for testosterone and membrane synthesis, respectively.