Isolation and characterization of a single cell suspension from the rat mammary gland was achieved by combining selective enzymatic digestion and the mechanical agitation of a Stomacher laboratory blender with immunohistological identification of cell-specific markers. Utilizing this procedure we were able to isolate single cell suspensions of high yield (10 to 15 X 10(6) cells/rat) and viability (greater than 98%) with a concurrent decrease in isolation time and the amount of proteolytic enzymes required. Five distinct cell fractions were isolated from the mammary gland cell suspension after banding on discontinuous Percoll gradients. These populations were characterized both before and after primary cell culture by a combination of histological, immunohistological, and autoradiographic techniques. Fractions two and three were found to be enriched for mammary epithelial cells, as identified by their high binding of antikeratin antibodies. These populations also exhibited a minimal degree of binding to actin, myosin, and fibronectin antibodies. Fraction three also exhibited a high labeling index as measured by autoradiography following in vivo administration of [methyl-3H]thymidine. The remaining fractions were found to contain higher percentages of myoepithelial cells or other mammary cell types. Inasmuch as there is a direct correlation between mammary gland cell types and susceptibility to mammary gland carcinomas, further studies of these cell populations may provide new insights into the mechanisms underlying mammary gland carcinogenesis.