We have recently demonstrated that the amiloride-sensitive Na+ channel in the apical membrane of the renal epithelial cell line, A6, is modulated by the alpha i-3 subunit of the Gi-3 protein. We also showed that a 700-kDa protein complex can be purified from the membranes of A6 epithelia which (a) can reconstitute the amiloride-sensitive Na+ influx in liposomes and planar bilayer membranes and (b) consists of six major protein bands observed on reducing sodium dodecyl sulfate-polyacrylamide gels with molecular masses ranging from 35 to 320 kDa. The present study was undertaken to determine if the alpha i-3 subunit was a member of this Na+ channel complex. G alpha i structure and function were identified by Western blotting with specific G alpha i subunit antibodies and Na+ channel antibodies, through ADP-ribosylation with pertussis toxin, and by immunocytochemical localization of the Na+ channel and G alpha i proteins. We demonstrate that two protein substrates are ADP-ribosylated in the 700-kDa complex in the presence of pertussis toxin and are specifically immunoprecipitated with an anti-Na+ channel polyclonal antibody. One of these substrates, a 41-kDa protein, was identified as the alpha i-3 subunit of the Gi-3 protein on Western blots with specific antibodies. Na+ channel antibodies do not recognize G alpha i-3 on Western blots of Golgi membranes which contain alpha i-3 but not Na+ channel proteins, nor do they immunoprecipitate alpha i-3 from solubilized Golgi membranes; however, alpha i-3 is coprecipitated as part of the Na+ channel complex from A6 cell membranes by polyclonal Na+ channel antibodies. Both alpha i-3 and the Na+ channel have been localized in A6 cells by confocal imaging and immunofluorescence with specific antibodies and are found to be in distinct but adjacent domains of the apical cell surface. In functional studies, alpha i-3, but not alpha i-2, stimulates Na+ channel activity. These data are therefore consistent with the localization of Na+ channel activity and modulatory alpha i-3 protein at the apical plasma membrane, which together represent a specific signal transduction pathway for ion channel regulation.