The tissue distribution and levels of estrogen receptor in neonatal mouse uterine tissue were determined in epithelial and stromal fractions separated by mild enzymatic treatment. Proteins of the isolated fractions were separated by gel electrophoresis and receptor was detected on immunoblots with monoclonal antibody H-222. Estrogen receptor protein was detectable in samples of reproductive tract tissue from 5- and 10-day-old mice. The level of receptor in 5-day-old animals was lower per unit DNA in epithelial cells than in stroma. Receptor levels were increased in both tissue types after treatment with diethylstilbestrol, but not with progesterone. Receptor protein present in these neonatal tissues was able to bind steroid as evidenced by affinity labeling with tamoxifen aziridine. Immunohistochemistry on sections of uteri from 4- and 10-day-old mice confirmed the biochemical results and indicated lower nuclear straining in epithelial cells than in stromal cells of uteri of 4-day-old mice. These results demonstrated that estrogen receptor protein is present in both epithelium and stroma of the neonatal mouse uterus, but at a higher level in stromal cells.