The layers of keratinocytes form an acid mantle on the surface of the skin. Herein, we investigated the effects of acidic pH on the membrane current and [Ca2+]c of human primary keratinocytes from foreskins and human keratinocyte cell line (HaCaT). Acidic extracellular pH (pHe≤5.5) activated outwardly rectifying Cl– current (ICl,pH) with slow kinetics of voltage-dependent activation. ICl,pH was potently inhibited by an anion channel blocker 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid (DIDS, 73.5% inhibition at 1 μ M). ICl,pH became more sensitive to pile by raising temperature from 24°C to 37°C. HaCaT cells also expressed Ca2+-activated Cl– current (ICl,Ca), and the amplitude of ICl,Ca was increased by relatively weak acidic pHe (7.0 and 6.8). Interestingly, the acidic pHe (5.0) also induced a sharp increase in the intracellular [Ca2+] (Δ[Ca2+]acid) of HaCaT cells. The Δ[Ca2+]acid was independent of extracellular Ca2+, and was abolished by the pretreatment with PLC inhibitor, U73122. In primary human keratinocytes, 5 out of 28 tested cells showed Δ[Ca2+]acid. In summary, we found ICl,pH and Δ[Ca2+]acid in human keratinocytes, and these ionic signals might have implication in pathophysiological responses and differentiation of epidermal keratinocytes.