Proliferation and differentiation in many cells are linked to specific changes in transmembrane ion fluxes. Previously, we have identified a nonspecific cation channel in keratinocytes, which is permeable to and activated by Ca++. To test whether this cation channel might serve as a pathway for Ca++ entry, we examined the effect of blocking this channel on membrane currents, markers of differentiation, and intracellular Ca++. In patch clamp studies, 10(-8) to 10(-6) M amiloride decreased the single-channel open probability. The same concentrations of amiloride inhibited the calcium-induced formation of cornified envelopes and activity of transglutaminase in a dose-dependent fashion. Amiloride inhibited the long-term rise of intracellular Ca++ induced by raised extracellular Ca++, without blocking the initial increase of intracellular Ca++. Amiloride at concentrations of 10(-7) to 10(-3) M did not change the resting intracellular pH of keratinocytes, although concentrations of 10(-6) M or greater inhibited the recovery from NH4(+)-induced acidification. To test whether the effect of amiloride was toxic, we measured DNA synthesis in the presence or absence of amiloride. DNA synthesis was unchanged, suggesting that amiloride's actions were not due to toxic effects. Although the exact mechanisms of amiloride's action remains to be determined, these experiments suggest that this compound may inhibit keratinocyte differentiation by blocking the nonspecific cation channel.