Recently we reported finding that prenatal exposure to a relatively low dose of 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) increases the rate of 7,12-dimethylbenz( a)anthracene (DMBA)-induced rat mammary carcinoma, while a high dose decreases it. One of the most important factors determining sensitivity of the mammary gland to neoplastic stimuli is its stage of differentiation at the time of exposure to the carcinogenic agent. Hence, to verify a biphasic dose-response relationship (enhancement of carcinogenesis at low dose, and inhibition at high dose), we investigated the effects of prenatal exposure to PCB126 on mammary gland differentiation. Female SD rats were injected (i.g.) with 25 pg, 2.5 ng, 250 ng, 7.5 μg of PCB126/kg, or the vehicle, on days 13–19 postconception. In 50-day-old offspring, regardless of the day of exposure to DMBA, only the 7.5 μg group showed statistically significant high levels of PCB126 in the fatty tissue of their mammary glands. Fifty-day-old female offspring of the 250 ng group showed apparent inhibition of the normal differentiation of terminal end buds (TEB) to alveolar buds and lobules (ABL), while those of the 7.5 μg group showed mammary gland hypoplasia. Expression levels of the estrogen receptor-α (ER) in TEBs and the ER mRNA in mammary glands were higher in the 7.5 μg, 250 ng, 2.5 ng groups. Proliferating cell nuclear antigen (PCNA) expression in TEBs of 50-day-old rats was statistically significantly higher in the 250 ng group and lower in the 7.5 μg group. In the developing mammary gland, TEBs are considered the most susceptible to mammary carcinogenesis, while ABLs are relatively protected from mammary carcinogenesis. Thus, prenatal exposure to a relatively low dose of PCB126 induced an alteration of mammary gland differentiation that might potentially increase the risk of DMBA-induced mammary carcinoma.