Alcohol abuse is a risk factor for cancers of the gastrointestinal tract, and it also can precipitate psoriasis characterized by hyperproliferation of epidermal cells. Because these effects of alcohol may involve stimulation of cell growth, and ethanol (EtOH) was shown to enhance DNA synthesis in mouse fibroblasts and epidermal cells, we conducted a study to determine whether EtOH can also stimulate mitogenesis in human fibroblasts and keratinocytes. In keratinocytes, EtOH had no effects on mitogenesis after shorter (17-hr) treatments, but it partially prevented inhibition of DNA synthesis elicited by longer treatments (3–4 days) with 2 mM calcium (Ca), a differentiation-inducing agent. In contrast, treatment of serum-starved zinc-treated (40 μM) human skin fibroblasts with 50–60 mM EtOH for 17 hr resulted in increased DNA synthesis. EtOH-induced DNA synthesis was blocked by 1 mM EGTA, a specific Ca chelator. Despite the presence of 1.8 mM Ca in the cell culture medium, the addition of 1 mM extra Ca (final concentration, 2.8 mM) for 17 hr induced DNA synthesis, presumably mediated by Ca receptors. In eight independent human skin fibroblast lines examined, treatment with EtOH for 46 hr, but not for 17 hr, invariably enhanced the effects of Ca on DNA synthesis, consistent with synergistic stimulation of cell proliferation by EtOH and Ca. Neomycin, a Ca receptor agonist, and EtOH also exerted synergistic effects on DNA synthesis after longer (46-hr) treatments. In mouse NIH 3T3 fibroblasts, both EtOH- and Ca-enhanced DNA synthesis after 17-hr treatment, but they stimulated cell proliferation only in combination. The results indicate that in human fibroblasts, EtOH can potentiate the longer-term effects of high concentrations of Ca on DNA synthesis whereas, in keratinocytes, EtOH may inhibit Ca-induced differentiation.