SUMMARY: Increased synthesis de novo of cholesterol by testes of rats and mice was found to be a contributing factor in the increased testicular cholesterol content after hypophysectomy. This was shown by increased [1-14C]acetate incorporation in vitro into free and esterified cholesterol by testicular tissue from rats and mice hypophysectomized 3 weeks earlier, as compared with intact controls. In mice, testicular cholesterol content was significantly increased 1 day after hypophysectomy at which time there was no change in the testis weight. Three weeks after the operation there was a significant decrease in testis weight and a significant increase in the concentration of free, esterified and total cholesterol as well as in the percentage of esterified fraction. These changes were even more pronounced 1 month later.