The peripubertal transition in male mammals is accompanied by a gradual decrease in sensitivity to the inhibitory effects exerted by gonadal hormones, such as T and E2. Here, we investigated the effects of chronic T and its metabolites, 5α-dihydrotestosterone and E2 on the hypothalamo-pituitary-gonadal axis at puberty. We also examined if T effects are distinct or mediated through its conversion to 5α-dihydrotestosterone or E2. Twenty-day-old male Siberian hamsters were sc implanted with a SILASTIC brand capsule containing varying doses of T, 5α-dihydrotestosterone, or E2. Several functional parameters of the hypothalamo-pituitary-gonadal axis were evaluated including hypothalamic GnRH concentration, pituitary and plasma FSH levels, pituitary FSH and LH mRNA, and testicular status. Our results showed that gonadal steroids inhibited puberty in a dose-dependent manner as evaluated by testes mass (undiluted steroid: T, 27 ± 3 mg; 5α-dihydrotestosterone, 18 ± 1 mg; and E2, 62 ± 4 mg relative to cholesterol-implanted controls, 510 ± 42 mg). Also, T decreased plasma FSH below detectable levels, but pituitary FSH concentration was unaffected (1.37 ± 0.16 ng/μg protein) while E2-treated hamsters had normal plasma FSH levels (3.5 ± 0.98 ng/ml) yet significantly lower pituitary FSH concentration (0.09± 0.04 ng/μg protein). These results showed that the pathways of T and E2 action on the hypothalamo-pituitary-gonadal axis are distinct.