The identification of guanine nucleotide binding proteins (G proteins) in guinea-pig tissues was assessed by the adenosine diphosphate-ribosylation of the α subunit by Bordetella pertussis toxin using [α32P] nicotinamide adenine dinucleotide as the substrate followed by sodium dodecyl sulphate - polyacrylamide gel electrophoresis and autoradiography. Three tissues (inferior colliculus, neuroblastoma cells, and the organ of Corti) contained G0α (39 kD), as well as Gi2α (40 kD) and Gi1α and/or Gi3α (41 kD). The stria vascularis and the VIIIth nerve contained mainly Gi2α, Gi1α and/or Gi3α, but G0α was barely detectable. A purified preparation of outer hair cells from the organ of Corti contained all three pertussis toxin substrates including G0α, with the Gi2α (40 kD) subunit being the most prominent. The immunocytochemical localization of the G0α subunit was determined by light microscopy after incubating isolated outer hair cells, Hensen cells and the stria vascularis with affinity-purified anti-G0α antibodies. In hair cells a positive reaction was observed along the plasma membrane and around the perimeter of the cuticular plate (zona adherens). Positive reaction was also observed within the infracuticular network extending from the cuticular plate towards the nucleus in outer hair cells. Finally, the base of the outer hair cells also contained G0α. However, it is likely that the G0α that is present in this cell region is not within the hair cell itself, but rather in nerve terminals which remained attached during dissection.