Cholesteryl ester hydrolase (CEH) (EC 3.1.1.13) activity was assayed in the 104,000 X g supernatant (S104) of rat and mouse testes and livers at various temperatures between 27 C and 44 C. The CEH activity in the testis dropped from 44 pmol [4-14C] cholesteryl oleate hydrolyzed/hr/mg protein to 14 pmol hydrolyzed/hr/mg protein (a 68% decrease) between testicular and abdominal temperatures (32 C and 37 C, respectively) in the rat. This decrease in activity is essentially a reversible phenomenon. CEH from the testis S104 was stabilized in 10 mM EDTA and was purified by HPLC size exclusion. These steps did not alter the temperature effect previously noted. The temperature effect on the testicular CEH was demonstrated in vivo by assaying the enzyme following unilateral cryptorchidism. The HPLC purification yielded 3 peaks of CEH activity from the testicular S104. The 28,000 MW peak was found to be temperature insensitive while the 70,000 and 420,000 MW peaks were temperature labile. The liver CEH of both species remained relatively constant over the range 32-37 C. CEH is a potential regulator of both steroidogenesis and membrane composition in the testis and its temperature lability may suggest a unique regulatory mechanism responsible for impaired spermatogenesis seen with elevated testicular temperatures.