Plasma membranes from rat cardiac ventricular tissue and insulin receptors partially purified by wheat-germ-agglutinin chromatography were subjected to direct photoaffinity labelling with [alpha-32P]GTP in order to elucidate the presence of insulin-receptor-coupled GTP-binding proteins. In plasma membranes three proteins have been identified that exhibit an enhanced photolabelling with the nucleotide in response to insulin. The apparent molecular masses of these proteins were found to be 56, 60 and 74 kDa. Photolabelling of partially purified insulin receptors showed the copurification of the 60-kDa species, whereas the 56-kDa and 74-kDa proteins could not be detected. Furthermore, the 60-kDa G-protein was found to be specifically co-immunoprecipitated with the insulin receptor. Incubation of insulin receptors with insulin increased the labelling of the 60-kDa band to 205 +/- 27% (n = 5) of control. Immuno- and ligand-blotting experiments revealed the additional presence of a 39-kDa G(o)-like protein and two G-proteins with molecular masses of 24 and 26 kDa in the receptor preparation. Under basal conditions the insulin receptor and the 60-kDa G-protein exhibited an apparent inverse distribution between plasma and microsomal membranes with the G-protein being extensively labelled in the microsomal fraction. In conclusion, our data show that, in its native environment, the cardiac insulin receptor couples to at least three GTP-binding proteins. Out of these, a 60-kDa species of microsomal origin, copurifies with the insulin receptor. It is suggested that this G-protein is associated with the insulin receptor and may be involved in insulin receptor signalling in target cells.