Gonadotroph adenomas may exhibit qualitative and quantitative defects in gonadotropin biosynthesis and secretion. Hypersecretion of immunoreactive FSH dimers by these adenomas occurs frequently; however, it has not been known whether this FSH is biologically active. Using the granulosa cell aromatase bioassay and a highly specific immunoradiometric assay for FSH, we studied the serum bioactivity and bio- to immunoactivity (B/I) ratios of 14 men with FSH-secreting adenomas and compared these values to those of 11 age-matched normal men. In addition, three adenoma patients received TRH (400 μg, iv). The mean basal serum FSH level (international units per L), as measured by both bio- and immunoassays, and the FSH B/I ratios were significantly higher (P < 0.02, by Kolmogorov-Smirnov test) in the adenoma patients than in normal men (mean ± sem; adenoma patients: bioactivity, 68.8 ± 10.4; immunoreactivity, 34.8 ± 13.7; B/I ratio, 3.4 ± 0.6; normal men: bioactivity, 5.8 ± 1.2; immunoreactivity, 6.4 ± 0.8; B/I ratio, 0.90 ± 0.1). Both bio- and immunoactive FSH rose after TRH injection, resulting in maintenance of the B/I (mean ± SEM; pre-TRH: bio-FSH, 63.7 ± 22.4; immuno-FSH, 28.0 ± 14.1; B/I ratio, 2.8 ± 1.2; post-TRH: bio-FSH, 125.6 ± 42.7; immuno-FSH, 45.8 ± 21.8; B/I ratio, 3.5 ± 1.6). When gonadotroph adenoma cells from three separate patients were cultured and their conditioned media (n = 3) studied, relatively large amounts of both bio- and immuno-FSH were detected. Furthermore, the major isoelectric profile of bio-FSH (pH 4.9–3.0) in the conditioned medium from two such adenomas was shown by chromatofocusing to be comparable to that of purified human pituitary FSH (pH 5.2–3.6). We conclude that gonadotroph adenomas in men secrete FSH that is biologically active, both basally and in response to TRH.