These studies were undertaken to determine the relative importance of plasma lipoproteins and trophic hormones as regulators of cholesterol synthesis in 3 endocrine glands of the rat. In rats treated with 4-aminopyrazolo [3, 4-d] pyrimidine, the plasma cholesterol level was reduced from 56 to 5 mg/dl and this resulted in a decrease in cholesterol ester content in the adrenal gland from 26.26 to 1.12 mg/g and in the ovary from 4.61 to 2.54 mg/g but no change in the testis. The rates of sterol synthesis were markedly increased in adrenal gland (42-fold) and ovary (2.7-fold) but not in testis. In the presence of normal levels of endogenous plasma lipoproteins administration of adrenocorticotropin (ACTH) gel did not alter the rates of sterol synthesis in the adrenal gland but did essentially double the tissue content of cholesterol esters. In contrast, the administration of chorionic gonadotropin increased the rate of sterol synthesis in the ovary 4.1-fold while decreasing the cholesterol ester level from 7.32 to 1.15 mg/g and increased the rate of synthesis in the testis 10.4-fold under circumstances where there was no decrease in ester content. In the ovary the effect of the simultaneous administration of 4-aminopyrazolo [3, 4-d] pyrimidine and chorionic gonadotropin was additive, resulting in a 6-to 7-fold increase in the rate of sterol synthesis and a still further decrease in tissue cholesterol ester content. In 4-aminopyrazolo [3, 4-d] pyrimidine-treated animals, the continuous infusion of 20 mg of cholesterol carried in high density lipoprotein over 42 hr increased the cholesterol ester content of the adrenal gland from 1.4 to 18.8 mg/g and suppressed sterol synthesis from 381 to 29 nmol/g/h. In contrast, the infusion of an identical amount of cholesterol carried in low density lipoprotein caused only a slight increase in the cholesterol ester content and a modest suppression of synthetic activity. Similar results were found in ovary and testis. Finally, in these same groups of rats the increase in the plasma corticosterone, progesterone and testosterone levels was 6-to 12-fold higher with infusion of high density lipoprotein than with low density lipoprotein. It is concluded that lipoprotein cholesterol, rather than cholesterol newly synthesized in the glands, is the major substrate for the production of steroid hormones in the adrenal gland, ovary, and testis of the rat. Furthermore, these 3 endocrine glands preferentially take up and utilize high density lipoprotein cholesterol rather than low density lipoprotein cholesterol.