Leptin, a member of the cytokine family, increases at the onset of puberty, and its testicular receptors are functionally expressed during puberty. Previous studies showed an inhibitory effect of leptin on testosterone (T) production by adult but not prepubertal (<31 days old) rat Leydig cells when stimulated by hCG or 8-Br-cAMP. We studied the influence of leptin on T output at the onset of puberty (40 d.o.) and in adult (60 d.o.) rats. Leptin (1 nM) was added to purified Leydig cells along with hCG (50 ng/ml) or 8-Br- cAMP (20 ug/ml). In adult rats there was a 15% inhibition of hCG stimulated T output (59.4 ± 1.2 ng/ml versus 70.2 ± 6.2 ng/ml, p >0.05), whereas in 40 d.o. Leydig cells T was reduced by 20 % (4.92 ± 0.23 versus 6.1 ± 0.35 ng/ml, p = 0.01). In both adult and 40 d.o. rats, 8-Br-cAMP stimulated T output was more inhibited by leptin, suggesting inhibition downstream of c-AMP generation. We also looked at the effect of pretreatment with leptin (15, 30 and 60 minutes) before hCG stimulation. In the peripubertal rats inhibition was greater at all 3 points than when cells were exposed to leptin and hCG simultaneously. This profound inhibitory effect may be due to “desensitization” to subsequent hCG stimulation. Using ³H progesterone as precursor we also show that inhibition by leptin in peripubertal rats results from negative regulation of lyase activity of the CYP17 enzyme. In conclusion, our findings support the concept that leptin has a modulating effect on testosterone secretion at the peripubertal stage.