The sequence of the 350 amino acids in the alpha subunit of GTPase of bovine rod outer segments has been determined. Enriched GTPase mRNA was used to prepare a cDNA library in the expression vector lambda gt11 and several overlapping cDNA clones corresponding to the alpha subunit of the GTPase were identified. The cDNA sequence determined contains 93 nucleotides upstream of the 5' end of the coding region, 1050 nucleotides that specify the amino acid sequence, and 45 nucleotides downstream from the 3' end. The previously described partial amino acid sequences and the sequences at the ADP-ribosylation sites for cholera and pertussis toxins are all confirmed and fitted into the present complete sequence. Homologies are found between the sequence of the alpha subunit and those of other guanine nucleotide-binding proteins, the ras proteins, peptide chain elongation factors EF-Tu and EF-G, and the initiation factor IF2.