Cultured BALB/c mouse mammary gland epithelial cells of varying oncogenic potential in vivo have been examined for their ability to multinucleate in the presence of cytochalasin B (CB). Highly tumorigenic cell lines derived from mammary tumors with hormonal, viral, or chemical carcinogen etiologies were extensively multinucleated when cultured in CB-supplemented medium. Normal mammary gland cells from either pregnant or lactating animals were predominantly mono- or binucleate under comparable conditions. At low passage levels after cloning, cell lines derived from a chemical carcinogen-induced mammary tumor were weakly oncogenic and remained largely mono-, or binucleated when cultured in CB-supplemented medium. At higher cell passage levels, both the ability to produce tumors in vivo and the degree of multinucleation in CB-supplemented medium increased dramatically with the clonal cell lines. Thus, the response of cultured mouse mammary gland epithelial cells to CB in vitro may be useful as a marker of the oncogenic potential of such cells.