Male hormonal contraceptive regimens function by suppressing gonadotropin secretion, resulting in a dramatic decrease in testicular androgen biosynthesis and spermatogenesis. Animal studies suggest that persistent intratesticular (iT)-androgen production has a stimulatory effect on spermatogenesis in the setting of gonadotropin suppression. We hypothesized that men with incompletely suppressed spermatogenesis (>1,000,000 sperm/mL) during male hormonal contraceptive treatment would have higher iT-androgen concentrations than men who achieved severe oligospermia (<or=1,000,000 sperm/mL). Twenty healthy men ages 18-55 years enrolled in a 6-month male contraceptive study of transdermal testosterone (T) gel (100 mg/d) plus depomedroxyprogesterone acetate (300 mg intramuscularly every 12 weeks) with or without the gonadotropin releasing hormone (GnRH) antagonist acyline (300 microg/kg subcutaneously every 2 weeks for 12 weeks) were studied. During the 24th week of treatment, subjects underwent fine needle aspirations of the testes and iT-T and iT-dihydrotestosterone (iT-DHT) were measured in testicular fluid by liquid chromatography-tandem mass spectrometry. All men dramatically suppressed spermatogenesis; 15 of 20 men were severely oligospermic, and 5 of 20 suppressed to 1.5 million-3.2 million sperm per milliliter. In all subjects, mean iT-T and iT-DHT concentrations were 35 +/- 8 and 5.1 +/- 0.8 nmol/L. IT-androgen concentrations did not significantly differ in men who did and did not achieve severe oligospermia (P = .41 for iT-T; P = .18 for iT-DHT). Furthermore, there was no significant correlation between iT-T or iT-DHT and sperm concentration after 24 weeks of treatment. In this study of prolonged gonadotropin suppression induced by male hormonal contraceptive treatment, differences in iT-androgens did not explain differences in spermatogenesis. Additional studies to identify factors involved in persistent spermatogenesis despite gonadotropin suppression are warranted.