The use of gene transfer procedures has greatly facilitated the investigation of cell lineage relationships and other developmental processes in a variety of primary tissues. In this report we described the infection and selection of primary human breast epithelial cells using retroviral vectors (Jzen-HSA-NEO and MSCV-HSA.NEO) containing the complete 228 bp coding sequence of a murine cell surface marker (Heat Stable Antigen, HSA) as well as the neomycin resistance (neo(r)) gene. Expression of the transduced HSA gene was detectable using either flow cytometry or immunohistochemistry after staining cells with an anti-murine HSA-specific antibody (M1/69). Expression of the transduced neo(r) gene conferred resistance to G418. In initial experiments with the MCF-7 breast cancer cell line, continued expression of both markers was demonstrated in a high proportion of cells for at least 4 weeks after their infection by positive M1/69 staining of cells that had been selected by prior incubation in G418. Evidence of gene transfer to early stage (< 9 days old) primary cultures of normal human breast epithelial cells (15 experiments with cells from 12 normal individuals) was also obtained using an infection protocol in which these calls were exposed to helper-free viral supernatants (2 incubations, 4 to 6 hr each) after being subcultured for 12 to 18 hr to increase their rate of proliferation. The presence of 5-50% (mean = 26%) HSA+ cells was demonstrated in these experiments within 5 days after their infection and the HSA+ populations included both myoepithelial and luminal phenotypes. The transduced (HSA+) cells within both of these subpopulations could also be separately isolated by FACS and subcultured. These results should provide an important starting point for future studies of genetically modified or marked primary human breast epithelial cell populations.