The majority (87%) of N-nitrosomethylurea-induced rat mammary tumors regressed within 1 week after hypophysectomy (hypox). After a hypox-induced tumor regression, ovine PRL (oPRL), and 17 beta-estradiol (E2) were administered separately or in combination in order to define the individual role of these hormones in regulating tumor growth and influencing estrogen (E), progesterone (Pg), and PRL receptor (R) levels. Administration of E2 (2.5 micrograms twice daily) or oPRL (20 IU daily, started 5 days after hypox and continued for 10 days, resulted in stabilization of tumor growth. Simultaneous administration of E2 and oPRL resulted in a synergistic effect and reactivation of tumor growth. ER levels in mammary tumors were significantly lower than those in the control 15 days after hypox (P less than 0.01). Treatment with E2, oPRL, or both simultaneously had no significant effect on ER levels. A significant decline in PgR levels was noted at both 5 and 15 days after hypox. Whereas treatment with oPRL had no significant effect on PgR levels, E2 administration either alone or in combination with oPRL restored PgR levels to control values. PRLR levels were unchanged from control values at 5 days, but significantly declined (P less than 0.005) 15 days after hypox. Treatment with E2, oPRL, or both hormones simultaneously partially maintained PRLR and prevented the decline to the extremely low level noted in the untreated group. We conclude that the growth of nitrosomethylurea-induced rat mammary tumors is dependent on both E2 and PRL. There was a synergistic effect between E2 and PRL on tumor growth but not on ER, PgR, or PRLR. Neither E2 nor PRL had any significant effect on ER after hypox. PgR is under E2 control. Either E2 or PRL or both hormones were able to maintain PRLR in mammary tumors after hypox.