1. Microfluorometric Ca2+ measurements using FURA-2 and whole cell patch-clamp recordings were performed to investigate the Ca2+ permeability of ion channels underlying the proton-induced sustained cation current in adult rat dorsal root ganglion neurons. 2. In a subpopulation of these neurons, extracellular application of acidic solutions (pH 5.1) elicited a sustained cation current and a concomitant reversible rise in the intracellular free Ca2+ concentration ([Ca2+]i), which depended on the presence of external Ca2+. Ruthenium red (10 microM) reduced both the current and the rise in [Ca2+]i to about the same extent. 3. In the presence of 2 mM external Ca2+, sustained proton-induced currents reversed sign at -4.6 +/- 1.2 (SE) mV, with external Na+ and internal Cs+ as the major charge carriers. Increasing the external Ca2+ concentration to 30 mM shifted the reversal potential (Erev) by 3.0 +/- 0.9 mV toward more positive values, suggesting a permeability ratio of Ca2+/Cs+ of 0.41. A similar value (0.35) could be obtained from Erev (-21 mV) under bi-ionic conditions with 100 mM external Ca2+ and 154 mM internal Cs+. 4. These results demonstrate that the proton-activated cation channels investigated here are moderately permeable to Ca2+. This may lead to pathophysiologically relevant increases in [Ca2+]i on prolonged exposure of the cells to an acidic environment in inflamed or ischemic tissue.