To evaluate the role of gonadotropins in the control of Mullerian Inhibiting Substance (MIS) secretion, pregnant rats were injected with rabbit antiserum against luteinizing hormone releasing hormone (LHRH), and their pups replaced with luteinizing hormone (LH) and/or FSH. LHRH antiserum was given at 13 and 20 days of gestation. Male pups from mothers treated with LHRH antiserum were given 5 daily s.c. injections of FSH, LH, FSH and LH or vehicle. The male pups from mothers treated with normal rabbit serum were given vehicle s.c. Testicular fragments of 6-day old pups born to mothers treated with LHRH antiserum during pregnancy showed an increase relative to controls in MIS activity in a graded organ culture bioassay system (Grade 3.4±0.2 vs. 2.0±0.0) (p<0.001). FSH replacement given to pups from mothers treated with LHRH antiserum reduced testicular MIS secretion compared to vehicle treated pups from the same mothers (Grade 2.5±0.3 vs. 3.4±0.2) (p<0.05). Postnatal FSH replacement after immunologic blockade of gonadotropins in utero reduced MIS activity of the testes to the same level as was found in testes of 6-day control pups. In contrast MIS activity remained high despite postnatal LH replacement in pups born to mothers given LHRH -AS (Grade 3.5±0.4 vs 3.4±0.2). These studies suggest that secretion of MIS is dependent on normal hypothalamic secretion of LHRH and is under inhibitory FSH control.