The embryonic period during which Mullerian duct regression and Mullerian Inhibiting Substance (MIS) secretion occur was determined in canine embryos removed from timed pregnancies (32, 36, 37, 39, 42, and 46 days gestation). Sex chromosomes of each embryo were identified in metaphase spreads prepared from fibroblast cultures. Testicular differentiation, defined by seminiferous tubule formation and the presence of Sertoli cells and Leydig cells, and the degree of Mullerian duct regression were determined by careful morphologic analysis of histologic sections of canine embryonic gonads (n = 20) and Mullerian ducts (n = 20). MIS was detected immunohistochemically in embryonic testes using avidin-biotin complex enhancement of a specific rabbit polyclonal anti-MIS antibody. Testicular differentiation was observed at 36 days gestation. The earliest evidence of Mullerian duct regression in male embryos was observed at 36 days gestation, and regression was completed by 46 days gestation. Positive staining for MIS was present in testes from 36 to 46 days (n = 9). Staining was absent in the undifferentiated testis (n = 1) at 32 days gestation and in ovaries at all ages tested (n = 10). Thus, MIS is normally present throughout the critical period for Mullerian duct regression in the embryonic male dog.