Supra-pharmacological doses of the insulin analog X10 (AspB10) increased the incidence of mammary tumors in female Sprague-Dawley rats in chronic toxicity studies, most likely via receptor-mediated mechanisms. However, little is known about the expression of the insulin receptor family in the rat mammary gland. Using laser micro-dissection, quantitative RT-PCR and immunohistochemistry, we examined the expression of IR (insulin receptor), IGF-1R (IGF-1 receptor), IRR (insulin receptor-related receptor), ERα (estrogen receptor alpha), ERβ (estrogen receptor beta) and PR (progesteron receptor) in young, virgin, female Sprague-Dawley rats and compared to expression in reference organs. The mammary gland displayed the highest expression of IRR and IGF-1R. In contrast, low expression of IR transcripts was observed in the mammary gland tissue with expression of the IR-A isoform being 5-fold higher than the expression of the IR-B. By immunohistochemistry, expression of IR and IGF-1R was detected in all mammary gland epithelial cells. Expression of ERα and PR was comparable between mammary gland and ovary, whereas expression of ERβ was lower in mammary gland than in the ovary. Finally, expression of IGF-1R and PR in the mammary gland varied during the estrous cycle. These findings are important for the understanding of carcinogenic effects of insulin analogs in the rat mammary gland, and relevant for development of refined short-term models for preclinical safety assessment of insulin analogs.