Müllerian inhibiting substance (MIS) on rat Müllerian duct (Md), and four Müllerian-derived tumor cells: HeLa S-3, RL-95.2, A-431 and NIH:OVCAR-3 are recognized by the poly- and mono-clonal avian-MIS-antibodies (A-MIS-Abs) using avidin-biotin complex (ABC) immunolabeling techniques. Internalization of MIS-ligand complexes was successfully detected in HeLa S-3, OVCAR-3 and RL 95.2 cells. Control groups include: (i) the samples omitted primary antibody treatment, (ii) Wolffian duct by side of Md in the genital ridge, and (iii) another two MIS-negative tumor cell lines of non-Müllerian origin: Chang hepatoma ascites cell and mouse myeloma cell (X63-Ag 8.653). Genital ridges from rat embryos of 14d of gestation were removed under dissection microscope, fixed in 2.5% glutaraldehyde in D-PBS of pH 7.2 for 30 min, and sliced into 0.1-0.2 mm thick pieces. A-431, HeLa S-3 and NIH:OVCAR-3 were maintained in OPTI-MEM culture medium, RL-95.2 was cultured in F12 culture medium. The cells were transferred to 24-well flat bottom culture plates with Thermanox tissue culture coverslips. The immunolabeling of fixed and non-fixed samples were processed within the wells. These studies provide first immunocytochemical evidences for the similarity between A-MIS and M-MIS molecules by polyclonal and monoclonal A-MIS-Ab. It has also proved that the tumor cell lines, which were subjects of MIS inhibition of cell growth, showed MIS binding on cell surfaces.