The effects of crude lymphokine-enriched supernatants, purified recombinant lymphotoxin (LT), tumor necrosis factor-alpha (TNF), and gamma interferon (gamma IF) on proliferating human keratinocytes were assessed using two in vitro culture systems. Activated splenocyte supernatants inhibited keratinocyte colony growth on fibroblast feeder layers and arrested basal keratinocyte DNA synthesis within 24 h. Purified recombinant LT, TNF, and gamma IF inhibited cell proliferation in serum-free medium without noticeably affecting viability. Cytostasis was dose-dependent (up to 90% with LT or TNF and 99% with gamma IF) and was maximal within 24-36 h. Specific antibodies neutralized TNF- and gamma IF-mediated cytostasis. Combined treatment with LT (or TNF) and gamma IF increased the degree of cytostasis, particularly at low lymphokine concentrations. Maximum inhibition of DNA synthesis and the duration of exposure required for this inhibition were comparable for LT and TNF and differed for gamma IF. Each of these lymphokines induced cell enlargement, flattening, and vesiculation, with gamma IF apparently more potent in this respect than LT or TNF. Fusiform keratinocytes with diffusely distributed cytokeratin were observed after prolonged treatment with gamma IF alone or gamma IF plus either LT or TNF. Flow cytometric studies of lymphokine-treated keratinocytes indicated that LT, TNF, and gamma IF could enhance beta-2 microglobulin expression 1.5-fold to threefold, whereas only gamma IF induced class II antigens. Staining for class II and beta-2 microglobulin was reduced on cells treated with high concentrations of gamma IF compared with either optimally treated or untreated cells. The potential relevance of these findings to cutaneous immune defense and disease is discussed.