Using high-resolution Mono-Q anion-exchange chromatography, we purified four distinct GTP-binding proteins from bovine brain. Each consists of .alpha. and associated .beta./.gamma. subunits, and each is a substrate for pertussis toxin catalyzed ADP-ribosylation. We defined the relationship between the .alpha. subunits of the purified proteins and cloned cDNAs encoding putative .alpha. subunits (1) by performing immunoblots with peptide antisera with defined specificity and (2) by comparing the migration on two-dimensional gel electrophoreis of the purified proteins, and of the in vitro translated products of cDNAs encoding .alpha. subunits. Purified G proteins with .alpha. subunits of 39, 41, and kDa (G39, G41, and G40 in order of abundance) correspond to the product of G0, Gi1, and Gi2 cDNAs. We purified a novel G protein with .alpha. subunit slightly above 39 kDa (G39*). G39* is less abundant than G39, elutes earlier than G39 on Mono-Q chromatography, and has a more basic pI (6.0 vs 5.6) than G39. G39 and G39* however, are indistinguishable on immunoblots with a large number of specific antisera. The data suggest that G39* may represent a novel form of G0, differing in posttranslational modification rather than primary sequence.