2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mammary gland carcinogen present in the human diet. Herein, the expression of estrogen receptor alpha (ERalpha), estrogen receptor beta (ER beta) and progesterone receptor (PR) was examined in mammary gland carcinomas induced by PhIP in female Sprague-Dawley rats. Quantitative real-time polymerase chain reaction demonstrated that ER alpha, ER beta and PR were statistically elevated by 3-, 4- and 8-fold in carcinomas compared with normal mammary glands. By immunohistochemistry, carcinomas showed statistically higher nuclear expression of all three steroid receptors with the majority of carcinomas showing at least 10% of epithelial cells stained for ER alpha (49/55, 89%), ER beta (41/55, 75%) and PR (48/55, 87%). Furthermore, the level of expression of the three steroid hormone receptors was positively correlated with each other across the bank of carcinomas (Spearman analysis, P < 0.05). The expression of ER alpha in carcinomas was associated with tumor grade, extent of nuclear pleomorphism and cellular proliferation as measured by proliferating cell nuclear antigen (PCNA) and phospho-Rb immunostaining (Spearman analysis, P < 0.05). Confocal microscopy was used to measure the percentage of epithelial cells showing nuclear colocalization of receptors, PCNA, and cyclin D1. Colocalization of the receptors, and the colocalization of the receptors with PCNA and cyclin D1 was strikingly higher in carcinomas than in the normal mammary gland. In carcinoma cells, 37% of ER alpha positive epithelial cells were colocalized with PCNA in contrast to just 0.25% of cells in the normal mammary gland. The findings from this study indicate that ER alpha, ER beta and PR were co-upregulated and nuclear localized in epithelial cells from rat mammary carcinomas compared with normal mammary glands, and that the co-upregulation was positively correlated with proliferation and cell cycle progression in carcinomas.