A combination of microspectrofluorimetry and single cell voltage-clamp was used to examine the response to ATP of cultured neurons from rat dorsal root ganglia. ATP activated an inward current and a rise in internal calcium concentration that was dependent on the external calcium concentration and on the magnitude of the ATP-induced current response. The response was not affected by prerelease of internal calcium stores with caffeine. The rise in internal calcium was increased at hyperpolarized membrane potentials as the calcium driving force was increased. These results demonstrate that the ATP-gated channels in these cells can admit a significant amount of calcium in a physiological calcium gradient. This alternative calcium entry pathway could provide an internal calcium signal that is spatially distinct to that generated by voltage-gated calcium entry.