Calcium-induced keratinocyte differentiation is associated with tyrosine phosphorylation of a p62 protein which associates with the ras-GTPase activating protein (GAP). We have examined the nature of the calcium signal triggering p62 phosphorylation. EGTA, a specific chelator of calcium, was able to completely block calcium-induced p62 phosphorylation, even after using conditioned medium from calcium-treated keratinocytes. Preventing calcium-induced cell-cell contacts by anti-cadherin antibodies did not inhibit tyrosine phosphorylation. Slight increases in extracellular calcium concentrations (0.15 or 0.30 mM) were already sufficient to induce p62 phosphorylation. Other divalent cations, such as magnesium, zinc, nickel, and cobalt, but not the trivalent cation lanthanum, induced p62 phosphorylation to a similar extent as calcium. There was no close correlation between the ability of the various ions to induce p62 phosphorylation and increase free intracellular calcium. Similarly, treatment of primary keratinocytes with the calcium ionophores A23187 or X537A did not induce p62 phosphorylation, although it increased free intracellular calcium levels. Finally, blockers of potassium uptake, which is induced by calcium, did not inhibit p62 phosphorylation. Thus, in keratinocyte differentiation, calcium is likely to provide the primary signal for p62 tyrosine phosphorylation and may act directly at the cell membrane through a "cationic receptor mechanism" analogous to that described in other cell types.