To determine the susceptibility of mammary epithelial cells (MEC) to HIV-1 as breastfeeding is an established route of HIV transmission, although the origin of virus in breastmilk is unclear. Primary epithelial cell cultures were derived from the mammary glands of healthy donors; immortalized MEC lines were also used. HIV infection was followed by detection of infectious particle production, p24 antigen and viral sequences. Seven out of 11 primary MEC cultures and two out of three MEC lines were productively infected by HIV-1. Virus replication significantly reduced cell proliferation, although cell viability was only slightly affected. Cytopathic changes were not observed. MEC cultures expressed low levels of surface CD4, galactosylceramide and CD26, but essentially no human leukocyte antigen (HLA)-DR. Infection of HIV-permissive MEC cells was associated with the upregulation of surface HLA-DR and CD26. In contrast, the expression of CD4, tissue-specific markers, adhesion molecules and growth-factor receptors was downregulated. To a lesser extent, similar effects were also observed in non-permissive cells. Hormones (triiodothyronine plus beta-estradiol and prolactin) enhanced HIV replication, possibly through the stimulation of cellular DNA synthesis. We concluded that HIV-1 replication in ductal/alveolar MEC may be, in part, responsible for the presence of HIV-1 in milk; that hormones may stimulate virus replication; and that infection reduces the growth of epithelial cells. Although in vitro HIV is produced by MEC to a lesser extent than lymphoid cells, MEC-derived HIV might have selective advantages for the infection of mucosal epithelial cells during breastfeeding.