[Ca2+]i and pH have been demonstrated to affect Na+ transport in epithelium mediated via the apical epithelial Na+ channel (ENaC). However, it still remains unclear whether the effects of [Ca2+]i and intracellular pH (pHi) on ENaC activity are direct. In this study, inside-out recording was employed to clarify the effects of pH(i) and [Ca2+]i on ENaC activity. We found that elevation of [Ca2+]i induced a significant inhibition of ENaC open probability without altering channel conductance. The inhibitory effect was due to a direct interaction between Ca2+ and ENaC, and is dependent on [Ca2+]i. pHi also directly regulated ENaC open probability. Lower pHi (7.0) enhanced the ENaC open probability as shown in augmented opening time. pHi did not cause any alteration in channel conductance. The effects of pHi on ENaC open probability could be summarized as an S-shaped curve around pH 7.2.