Synthetic progestins such as levonorgestrel (LNG) are used in combination with testosterone (T) in male contraceptive clinical trials to suppress gonadotropins secretion, but whether progestins have additional direct effects on the testis are not known. This study aimed to examine the effect of a potent progestin, (LNG), alone or in combination with testosterone (T) on spermatogenesis in adult rats, and to evaluate the functional role of the progesterone receptors (PRs) in the testis. In comparison with a low dose of LNG treatment in adult rats for 4 weeks, T and T + LNG treatment decreased testicular sperm count to 64.1 and 40.2% of control levels respectively. LNG induced germ cell apoptosis at stages I–IV and XII–XIV; T increased apoptosis at stages VII–VIII; LNG + T treatment induced greater germ cell apoptosis at a wider range of seminiferous epithelial stages. RT-PCR and Western Blots showed that PR was present in testes and up-regulated during suppression of spermatogenesis induced by testicular hormonal deprivation. PR knockout (PRKO) mice had larger testes, greater sperm production, increased numbers of Sertoli and Leydig cells. Suppression of gonadotropin and intratesticular T by GnRH-antagonist treatment induced PR promoter driven LacZ expression in Leydig cells of PRKO mice. This suggests that GnRH-antagonist treatment while inducing germ cell apoptosis also up-regulates PR. We conclude that (i) LNG + T induced greater suppression of spermatogenesis through increase in germ cell apoptosis involving a wider range of seminiferous epithelial stages than either treatment alone, (ii) up-regulation of PR was associated with inhibition of spermatogenesis, (iii) PR knockout mice showed increased sperm production suggesting that testicular PR activated events play a physiological and pharmacological inhibitory role in the testis. These data support the hypothesis that in addition to its known suppressive effects on gonadotropins, progestins may have direct inhibitory actions on the testis.