The activities of a lysosomal acid and two neutral cholesteryl ester hydrolases (microsomal and cytosolic) found in the mammary gland were studied in subcellular fractions prepared from tissue from rats at various stages of pregnancy, lactation, and after weaning the pups. The relationship between the activities of these enzymes and that of acyl coenzyme A: cholesterol acyl transferase (ACAT) was also investigated. Acid cholesteryl ester hydrolase activity was increased considerably in glands from lactating as compared to pregnant animals, and was sharply decreased 2 days after weaning. The microsomal neutral cholesteryl ester hydrolase activity followed a similar pattern, but took longer to return to pre-lactating values on weaning. In contrast, the activity of the cytosolic neutral cholesteryl ester hydrolase increased throughout pregnancy, then remained relatively constant in lactation and for 2 days after weaning. At 8 days post-weaning, however, activity was markedly decreased. No correlation between the changes in the activities of any the cholesteryl ester hydrolases and ACAT during mammary gland development was detected. The microsomal neutral cholesteryl ester hydrolase activity showed a strong positive linear correlation with the unesterified and esterified cholesterol content of the microsomal fraction. No relationship, however, was found between the cytosolic neutral cholesteryl ester hydrolase and the level of either form of cholesterol in the cytosol. In addition, there was no correlation between ACAT activity and microsomal cholesterol concentrations. These results provide evidence for an important role for the enzymes responsible for cholesteryl ester synthesis and hydrolysis in the mammary gland in the regulation of cholesterol metabolism and the provision of cholesterol for secretion into milk.