Cell lines and sublines initiated from BALB/cfC3H mammary adenocarcinomas exhibited varying morphologies during the 15- to 21-month culture periods. Two epithelial cell lines, FUKU and MJY, continued to form the hemicysts and mounds characteristic of primary cell cultures. Subline MJY-alpha did not form these structures. Instead, the cells varied from polygonal to fusiform within each passage level and, when confluent, the monolayers continually released viable cells into the culture fluid. When introduced into isogeneic hosts, FUKU rapidly established adenocarcinomas with acinar areas, though MJY-alpha yielded only slow-growing carcinosarcomas. 3H-uridine incorporation revealed the constant release of virions from the cell lines and subline. Immunodiffusion assays of virus purified from the culture fluids indicated that FUKU released a mixture of virions containing both mammary tumor virus (MTV) and murine leukemia virus (MuLV) antigens; MJY-alpha virions, on the other hand, were positive for only MTV antigens. Electron microscopic examination of the cell lines and purified virions substantiated this finding. Immunofluorescence detected a shift in the localization of MTV antigens in fixed cell layers of long-term cultures. With increasing passage levels, the cytoplasmic fluorescence typical of primary cultures was replaced by a cell membrane-associated staining pattern. By the fourth to sixth passage level, only cell-surface fluorescence was observed with use of anti-MTV antisera.