A newly developed culture system was used to examine the proliferative potential of rat mammary epithelial (RME) cells in vitro. RME cells were obtained by enzymatic dissociation of mammary tissues of 45- to 50-year-old virgin female LEW rats. The tissues were dissociated to small aggregates (10-50 cells per aggregate) separated from stromal cells and plated at a density of 10(5) cells per 60-mm tissue culture dish. The cells were grown in Ham's medium F12 supplemented with 5% fetal bovine serum, insulin, hydrocortisone, epidermal growth factor, prolactin, progesterone, and cholera toxin. Plating of 10(5) cells as small aggregates resulted in the attachment of 1,000-1,500 aggregates per plate. When grown on tissue culture plastic, approximately 1-2% of these aggregates gave rise to rapidly proliferating epithelial colonies. Individual colonies expanded with a population-doubling time of 24-34 hours and grew for about 3 weeks. Although these cells grew well in primary culture, they were not subculturable. When RME cells were plated onto dishes coated with type I collagen, the number of rapidly proliferating epithelial colonies per dish increased fivefold to tenfold. Cells grown on type I collagen-coated dishes expanded with a population-doubling time of approximately 27 hours and after 2 weeks in primary culture were nearly confluent. Unlike cells grown on plastic, RME cells grown on type I collagen were readily subculturable and serial subculture resulted in the cells undergoing 15-20 population doublings (5-6 passages) before exhibiting any loss of growth potential. Continued feeding of senescent cultures resulted in the emergence of discrete RME cell foci that retained proliferative potential and that eventually developed into rapidly growing cell strains. Exposure of primary cultures to the carcinogen N-methyl-N'-nitro-N'-nitrosoguanidine (CAS: 70-25-7) enhanced the proliferative potential of RME cells in early passages and in later passages either delayed or eliminated the "senescent" phase of cell growth. Carcinogen treatment of RME cells also facilitated the establishment of rapidly growing cell strains with long-term growth potential (greater than 20 passages).