Cholesteryl ester hydrolase (CEH) activities with different pH optima, i.e. at about pH 4.0 and pH 7.0, were found in Leydig cells isolated from rat testis. Highly purified intact Leydig cells responded to human chorionic gonadotropin treatment with increased intracellular cAMP levels, increased testosterone release, and a 1.5-fold stimulation of the acid CEH activity. Treatment of non-stimulated homogenised cells with cAMP-dependent proteinkinase, cAMP and ATP lead to a 3.0-fold stimulation of the acid CEH activity. These findings suggest that interconvertible CEH with an acidic pH optimum--presumably of lysosomal location--contributes to the regulation of steroidogenesis in Leydig cells.