Mullerian inhibiting substance (MIS) was studied in the testis, ovary, and ovotestis of the domestic fowl, from early embryonic development to adulthood by a graded organ-culture method using the urogenital ridge of a 14.5 day fetal rat. The chick testis had high levels of MIS from 8 days of embryonic development (4-5+) until sexual maturity, when activity declined (2+) but persisted throughout adulthood. The right gonad (ovotestis) of the hen showed moderate levels of MIS activity from 8 to 12 days of embryonic life (1-3+) before its normal involution by the time of hatching (21 days). The left gonad (ovary) of the hen also produced MIS at levels similar to that seen in the testis. Activity was high during embryonic development (4+) and after hatching, and persisted in the adult hen at diminished levels (2+). MIS production by the female gonads explains regression of the right Mullerian duct, but the mechanisms that allow preservation of the left duct remain unexplained. Evidence suggests that ovarian estrogens may interact with MIS at the receptor level to protect the left Mullerian duct. MIS persists in the sexually mature gonads of both the hen and cockeral, long after the Mullerian duct has regressed, suggesting a wider role for this substance in reproductive physiology.