Despite being widely used to investigate 17β-estradiol (E2)-induced mammary gland (MG) carcinogenesis and prevention thereof, estrogen homeostasis and its significance in the female August Copenhagen Irish (ACI) rat model is unknown. Thus, levels of 12 estrogens including metabolites and conjugates were determined mass spectrometrically in 38 plasmas and 52 tissues exhibiting phenotypes ranging from normal to palpable tumor derived from a representative ACI study using two different diets. In tissues, 40 transcripts encoding proteins involved in estrogen (biotrans)formation, ESR1-mediated signaling, proliferation and oxidative stress were analyzed (TaqMan PCR). Influence of histo(patho)logic phenotypes and diet on estrogen and transcript levels was analyzed by 2-way ANOVA and explanatory variables influencing levels and bioactivity of estrogens in tissues were identified by multiple linear regression models. Estrogen profiles in tissue and plasma and the influence of Hsd17b1 levels on intra-tissue levels of E2 and E1 conclusively indicated intra-mammary formation of E2 in ACI tumors by HSD17B1-mediated conversion of E1. Proliferation in ACI tumors was influenced by Egfr, Igf1r, Hgf and Met levels. 2-MeO-E1, the only oxidative estrogen metabolite detected above 28-42 fmol/g, was predominately observed in hyperplastic tissues and intra-tissue conversion of E1 seemed to contribute to its levels. The association of the occurrence of 2-MeO-E1 with higher levels of oxidative stress observed in hyperplastic and tumor tissues remained equivocal. Thus, the present study provides mechanistic explanation for previous and future results observed in the ACI model.