Regression of the fetal rat Mullerian duct in vitro was stimulated by sodium fluoride in the absence of Mullerian inhibiting substance. The action of Mullerian inhibiting substance was inhibited by sodium vanadate, adenosine 5'-triphosphate, and several related nucleotides in the presence of manganese ions. Epidermal growth factor specifically inhibited the substance, but only with manganese ions present. Insulin, platelet-derived growth factor, and nerve growth factor had no effect. These results suggest that dephosphorylation of membrane proteins mediates the action of Mullerian inhibiting substance.