The MTW9/PL cell line was established by our laboratory in culture from the carcinogen-induced hormone-responsive MT-W9A rat mammary tumor of a Wistar–Furth (W/Fu) rat. This tumor formed estrogen, androgen, and progesterone responsive tumors in W/Fu rats (Sirbasku, D. A., Cancer Res. 38:1154–1165; 1978). It was later used to derive the MTW9/PL2 cell population which was also estrogen-responsive in vivo (Danielpour, D., et al., In Vitro Cell. Dev. Biol. 24:42–52; 1988). In the study presented here, we describe serum-supplemented culture conditions in which the MTW9/PL2 cells demonstrate ≥80-fold steroid hormone growth responses. All sera used were steroid hormone–depleted by charcoal–dextran treatment at 34° C. The studies were done with horse serum as well as serum from other mammalian species. The growth of the MTW9/PL2 cells was biphasic in response to hormone-depleted serum. Concentrations of ≤5% (v/v) promoted optimum growth. Above this concentration, serum was inhibitory. Concentrations ≥40% (v/v) inhibited growth altogether. Addition of 1.0 × 10⁻¹³–1.0 × 10⁻⁸ M 17β-estradiol (E₂) reversed the inhibition completely. At 1.0 × 10⁻⁸ M, estrone, estriol and diethylstilbestrol promoted growth as well as E₂. Testosterone and dihydrotestosterone promoted growth only at ≥10⁻⁷ M. Progesterone was effective only at ≥10⁻⁶ M. Cortisol was ineffective. Labeled-hormone–binding analysis and Western immunoblotting documented that MTW9/PL2 cells had estrogen and progesterone receptors but not androgen or cortisol receptors. Estrogen treatment of MTW9/PL2 cells induced a concentration and time dependent increase in progesterone receptors. We conclude (1) the MTW9/PL2 population is the first highly steroid hormone–responsive rat mammary tumor cell line to be established in culture from a carcinogen-induced tumor, and (2) sera from a number of species including horse, rat and human contain an inhibitor which mediates estrogen sensitive MTW9/PL2 cell growth in culture.