Mullerian Inhibiting Substance (MIS) is a testicular product that causes the regression of the Mullerian duct in the developing male embryo. Antibody specific for MIS would facilitate the purification and study of this "hormone," but because of its impure status, traditional polyclonal antisera specific for MIS would be untenable. The requisite specificity, however, might be obtained by the technique of somatic fusion, regardless of the purity of the immunizing antigen. This paper describes the production of 2 monoclonal antibodies specific for MIS by the technique of somatic cell fusion. Spleen cells from mice immunized with an impure preparation of MIS were fused with myeloma cell line NS1. Culture media from the resulting hybridoma cell lines were screened for anti-MIS antibody by a sensitive RIA. Specificity for MIS was demonstrated by the adsorption of biologically active MIS on an affinity column prepared from monoclonal anti-MIS antibody. MIS activity as assessed by an organ culture assay was subsequently recovered from the affinity column in the fraction eluted with MH4SCN. Using the RIA, monoclonal anti-MIS antibody was also shown to compete favorably with a variety of potentially cross-reactive proteins.