We investigated the concentrations of serum inhibin B and the pro-αC-containing inhibins (pro-αC inhibin) and their relations to serum FSH levels in 38 healthy boys during their progression through puberty. Furthermore, we studied the effect of recombinant human FSH (rhFSH) on pro-αC inhibin production in three prepubertal gonadotropin-deficient boys. The serum inhibin B level increased between Tanner stages G1 and G2 (p < 0.02), simultaneously with the serum LH and testosterone concentrations. In advanced puberty, inhibin B changed less, and at stage G4, correlated negatively with serum FSH level (r = -0.57, p < 0.001, n = 37). The serum pro-αC inhibin level changed differently and increased also in advanced puberty. In prepubertal healthy subjects, the serum pro-αC inhibin and FSH levels correlated positively (r = 0.50, p = 0.051, n = 16), and during the rhFSH treatment of gonadotropin-deficient boys, serum pro-αC inhibin levels increased. These findings suggest that an increased production of inhibin B is an early event in puberty and that inhibin B can inhibin pituitary FSH secretion in the course of sexual maturation. Furthermore, they suggest that during puberty free inhibin (pro)-α-subunits, detected by the pro-αC inhibin assay, are present in large amounts and that FSH stimulates their production by prepubertal testes.