Antisera were raised in rabbits against the 40-kDa alpha subunit of bovine lung GTP-binding protein, which were identified as the alpha subunit of Gi2 (Gi2 alpha) by the analysis of the partial amino acid sequence. Antibodies were purified with a Gi2 alpha-coupled Sepharose column and then were passed through a Gi1 alpha-coupled Sepharose column to remove antibodies reactive also with 41-kDa alpha. Purified antibodies reacted with Gi2 alpha, but not with Gi1 alpha, Gi3 alpha, or Go alpha in an immunoblot assay. A sensitive enzyme immunoassay method for the quantification of Gi2 alpha was developed by using these purified antibodies. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The minimal detection limit of the assay was 1 fmol, or 40 pg. Samples from various tissues were solubilized with 2% sodium cholate and 1 M NaCl, and the concentrations of Gi2 alpha were determined. Gi2 alpha was detected in all the tissues examined in the rat. The highest concentration was found in platelets and leukocytes when the data were expressed as picomoles per milligram of protein. The spleen, lung, and cerebral cortex contained relatively high levels of Gi2 alpha. In the bovine brain, Gi2 alpha was distributed almost uniformly among the various regions. The concentrations of Gi2 alpha were constant in the rat brain throughout ontogenic development, in contrast with those of Go alpha which were markedly increased with age.