Estrogens regulate gene expression and cell proliferation in target tissues. In studies of estrogen-regulated gene expression, identification of appropriate housekeeping genes (HKGs), reference genes whose expression is not altered by treatment, is difficult. The goal of this study was to define HKGs unaltered by estrogen in the mouse uterus. Ovariectomized C57BL6 mice were dosed with 20 micrograms/kg ethinylestradiol and the uterus was collected at 6, 24, and 72 h later to bracket the biphasic time course of estrogen action in the rodent uterus. RNA was isolated, cDNA synthesized and equal amounts of cDNA were added to real-time PCR reactions. The expression of seven out of nine putative HKGs was altered by estrogen in the mouse uterus. Estrogen induced four gene expression profiles, expression of: (1) Actb and Hsp90ab1 were up-regulated early, (2) B2m and Gusb were up-regulated late, (3) Gapdh, Hprt1, and Ppia were up regulated at all time points, and (4) Rpl13a and 18srRNA were unaltered. This highlights the need to empirically determine the appropriate HKG for each experimental condition. Based on these results, we suggest using Rpl13a or 18srRNA as HKGs for xenoestrogen studies in the mouse uterus and as good candidates to test under different experimental conditions.