A patient with gynecomastia and a nonpalpable testicular Leydig cell tumor is presented. Estradiol and progesterone levels were elevated whereas serum testosterone was reduced. Following removal of the tumor the hormonal values returned to normal with reduction of gynecomastia. Tumor tissue was maintained in culture for 9 days during which high estradiol levels as well as lesser quantities of testosterone and progesterone were demonstrated in the culture medium. Elevation of progesterone supports a block of 17 alpha-hydroxylase activity caused by increased endogenous estradiol. This is the first case of a cultured feminizing testicular Leydig cell tumor in which hormone production was demonstrated. The freshly removed tumor as well as the cultured tissue material were studied by immunocytochemical methods and biochemical analysis of cytoskeletal proteins. These methods revealed vimentin as the exclusive type intermediate sized filament in both. This is the first demonstration of vimentin in a Leydig cell tumor, in agreement with its occurrence in normal Leydig cells.