The Ca(2+) affinity and permeation of the epithelial Ca(2+) channel (ECaC1) were investigated after expression in Xenopus oocytes. ECaC1 displayed anomalous mole-fraction effects. Extracellular Ca(2+) and Mg(2+) reversibly inhibited ECaC1 whole cell Li(+) currents: IC(50) = 2.2 +/- 0.4 microM (n = 9) and 235 +/- 35 microM (n = 10), respectively. These values compare well with the Ca(2+) affinity of the L-type voltage-gated Ca(2+) (Ca(V)1.2) channel measured under the same conditions, suggesting that high-affinity Ca(2+) binding is a well-conserved feature of epithelial and voltage-gated Ca(2+) channels. Neutralization of D550 and E535 in the pore region had no significant effect on Ca(2+) and Mg(2+) affinities. In contrast, neutralization of D542 significantly decreased Ca(2+) affinity (IC(50) = 1.1 +/- 0.2 mM, n = 6) and Mg(2+) affinity (IC(50) > 25 +/- 3 mM, n = 4). Despite a 1,000-fold decrease in Ca(2+) affinity in D542N, Ca(2+) permeation properties and the Ca(2+)-to-Ba(2+) conductance ratio remained comparable to values for wild-type ECaC1. Together, our observations suggest that D542 plays a critical role in Ca(2+) affinity but not in Ca(2+) permeation in ECaC1.