In addition to leukocytes and fibroblasts, the classic sources of human interferons (IFN), many other human cells are now known to be capable of producing IFN. Keratinocytes (KC) are abundant in the skin and provide the first line of defense against viruses and other noxious agents. Human KC are a potent source of cytokines and were implicated as forming IFN-like protein(s). We have investigated whether KC form IFN. We found that culture supernatants from unstimulated human KC did not contain detectable amounts of IFN-α or IFN-β. However, those from KC activated with the potent IFN inducer, polyriboinosinic:polriboycytidylic acid (poly rI:rC), had appreciable antiviral activity, which studies with neutralizing sera showed to be caused by IFN-β. In ELISA tests, we detected IFN-β protein in the supernatants but not IFN-α protein. Nevertheless, reverse transcription PCR showed that both IFN-α and IFN-β mRNA were upregulated in poly rI:rC-treated KC. The levels of these mRNA were also increased in KC exposed to ultraviolet B (UVB) irradiation, interleukin-lα (IL-1α), tumor necrosis factor-α (TNF-α), or lipopolysaccharide (LPS). These results show that IFN-β is among the cytokines secreted from human KC and, together with IFN-α, may have a role in host defense mechanisms in the skin.