Mouse epidermal cells can be grown as a proliferating monolayer in medium containing 0.02-0.1 mM calcium. Terminal differentiation of these cells with formation of cornified cells and cell death is induced by elevating calcium in the medium to greater than 0.1 mM. A variety of agents were studied as potential modifiers of this calcium-induced terminal differentiation. Other than calcium, no cation tested was active in inducing or preventing epidermal maturation. Modifiers of calcium or sodium fluxes, local anesthetics and protease inhibitors were also without effect. Modulators or analogues of cyclic nucleotides did not influence epidermal differentiation, and cyclic nucleotide levels did not change significantly in the first 10 min after increasing calcium. Effective inhibition of calcium-induced differentiation, as estimated by morphology, ultrastructure and cornified envelope formation, was seen with the divalent cation ionophore A23187 and the Na+K+ATPase inhibitor ouabain. The well-known effects of ouabain on intracellular sodium and potassium suggested the possible involvement of these ions in the program of calcium-induced epidermal maturation. The increase in medium calcium produced an elevation of both intracellular sodium and potassium within 12-24 hours. The calcium-induced increase in intracellular potassium appears to be the more relevant of these changes since the increase was blocked by both ouabain and A23187. Other inhibitors of calcium-induced differentiation, including harmaline, 8(diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) and low potassium medium, also blocked the rise of intracellular potassium. The five inhibitors had no consistent effect on intracellular sodium. Thus, elevated intracellular potassium may be necessary for the later stages of epidermal differentiation. However, neither ouabain nor A23187 affected the assembly of desmosomes, the earliest ultrastructural change noted after increasing medium calcium. This rapid change in cell-cell contact, beginning within minutes after calcium elevation, appears to be independent of changes in sodium and potassium, but may instead be modulated by increased calcium at the cell surface.