In order to study lipid synthesis in female hamster flank organs during the estrous cycle, glands were obtained from different animals during different phases of the cycle. Lipid metabolism of [U-14C]glucose was studied under in vitro conditions. The radioactive lipids formed, were extracted from the glands and quantified. An aliquot of the extract was submitted to TLC to isolate and identify the lipids formed. Lipid synthesis in the glands increased significantly during estrous compared with the diestrous phase, suggesting the influence of sex hormones on lipid metabolism in flank organs. The radioactive lipids isolated and identified were: phospholipids, cholesterol, glycerides, waxes, and cholesterol esters. The percentages of conversation from [U-14C]glucose to cholesterol esters were minor in extracts from glands of diestrous animals as compared to estrous and proestrous animals, whereas glycerides and waxes increased significantly in metestrous and diestrous animals. To verify and evaluate the role of estrogen and progesterone in lipid synthesis, three different groups of gonadectomized female hamsters were injected daily with estradiol, progesterone, and 5 alpha-progesterone. After treatments, glands were incubated with [U-14C]glucose to determine the incorporation of radioactive glucose into lipids under culture conditions. The radioactive lipids synthesized were subsequently extracted and identified. The results showed that estrogen had no effect on in vitro [U-14C]glucose incorporation into lipids by flank organs, compared to the vehicle, whereas progesterone and 5 alpha-progesterone increased radioactive lipid synthesis by the glands significantly (p < 0.05). The radioactive lipids isolated and identified from extracts of gonadectomized female hamster flank organs were: phospholipids, cholesterol, triglycerides, waxes, and cholesterol esters. Gonadectomy increased phospholipid synthesis and decreased that of monoglycerides, diglycerides, and waxes. The percentages of conversion from [U-14C]glucose to cholesterol esters were higher after progesterone and 5 alpha-progesterone treatments than in vehicle and estrogen-treatments. Our data indicate that progesterone and 5 alpha-progesterone-treatments increased the in vitro [U-14C]glucose incorporation into lipids in flank organs from gonadectomized female hamsters. Furthermore, the major lipid synthesized by glands under these stimuli were cholesterol fatty acids. Thus, progesterone and 5 alpha-progesterone alter the consistency of sebum from gonadectomized female glands.