We have cloned and characterized cDNA encoding α(i), the GTP-binding subunit of G(i), a protein that mediates hormonal inhibition of adenylate cyclase and hormonal regulation of other membrane functions. We have also identified cDNA encoding a putative protein, which we have named α(h), that is highly homologous to α(i) but different from other known GTP-binding proteins. Both cDNAs were isolated from a bovine pituitary library. The cDNA encoding α(i) was identified by finding that the amino acid sequence determined for two tryptic peptides from α(i) agreed exactly with amino acid sequences deduced from the cDNA. We also determined the amino acid sequence of peptides derived from α(o), a related 39-kDa protein purified from bovine brain. These sequences are ~ 75% identical to the sequence determined for α(i). Southern blot analysis of bovine genomic DNA, using as probes radiolabeled cDNAs for α(i), α(h), and the α subunit of a related protein, transducin, showed that each probe recognized different genomic DNA fragments. Our results suggest a further level of complexity in the organization of the G-protein gene family, with multiple G proteins of very similar structural properties likely to be identified as products of distinct genes.