A newly established cell line of a granulocytosis/hypercalcemia-inducing murine mammary carcinoma (CE mammary carcinoma) grown in serum-free culture medium secretes factors that stimulate proliferation of granulocytes and embryonal bone cells. These cultured cells retain the ability to produce granulocytosis and hypercalcemia when they are transplanted back into mice. In culture these cells form clusters of organized cells. Studies by scanning, transmission, and freeze-fracture electron microscope techniques reveal that these in vitro tumor cells retain the structural epithelial characteristics of mammary epithelia. They maintain cellular polarity, microvilli, and complete tight junctions. Both the in vivo and in vitro tumor cells produce viral particles with the ultrastructural features of murine mammary tumor viruses. In both in vivo and in vitro conditions, A-type particles are present intracellularly. B viral particles are present predominantly in the intercellular spaces. Since the structural characteristics of the cultured tumor cells are consistent with the features of mammary adenocarcinomas, this is a prime culture system for studying the tumor-derived soluble factors.