Prolactin activity in the sera of 29 patients with gynecomastia associated with a variety of clinical conditions has been assayed by a new and highly specific bioassay method, which relates the rate of 32P-casein formation by cultured mouse mammary cells to the concentration of prolactin. The lower limit of detection in this assay is 2 ng/ml (0.06 mU/ml) above normal control levels. Prolactin was not detectably elevated in any patients with gynecomastia except those being treated with tranquillizer drugs or reserpine. Elevated levels of chorionic somatomammotrophin were found in one patient with gynecomastia and testicular choriocarcinoma, although a similar level in a second patient with choriocarcinoma was not associated with gynecomastia. Although elevated prolactin levels may be an associated finding in some patients with gynecomastia, these results fail to provide evidence that prolactin is etiologically related to the development of this condition.