The epidermis is constantly exposed to a variety of microbial pathogens and plays a vital role in resisting them. Soluble CXC chemokine ligand (CXCL) 16, which is one of the ELR- CXC chemokines, acts as a mediator of innate immunity by attracting CXC chemokine receptor (CXCR) 6-expressing cells, such as activated T cells and NKT cells. However, the production of CXCL16 by non-immune cells remains unclear. We found that cultured keratinocytes produced a significant amount of CXCL16 (2-3 ng per 10(6) cells per 24 h). Stimulation with tumor necrosis factor alpha, IL-1alpha, IFN-gamma, peptidoglycan and polyinosinic-polycytidylic acid [poly(I:C)] enhanced CXCL16 production. The forms of CXCL16 in the culture supernatants had molecular weights of 14, 28 and 50 kDa. Immunohistochemical analysis revealed that the normal human epidermis expressed CXCL16. As several chemokines have anti-microbial activities, we studied the anti-microbial activity of CXCL16. The chemokine domain of CXCL16 at concentrations >5 microg ml(-1) had significant anti-microbial activity against Staphylococcus aureus and Escherichia coli. Killing activity was retained at the physiological salt concentration in the presence of carbonate. In conclusion, CXCL16 is a novel mediator of the innate immune reactivities of epidermal keratinocytes.