We have cloned the human chromosomal gene coding for the alpha subunit of Gx (Gx alpha), a heterotrimeric signal-transducing GTP-binding protein (G protein) that is insensitive to pertussis toxin. Gx alpha cDNA has been cloned both from rat brain (Matsuoka, M., Itoh, H., Kozasa, T., and Kaziro, Y. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5384-5388) and from human retina (referred to as Gz alpha, Fong, H.K.W., Yoshimoto, K.K., Eversole-Cire, P., and Simon, M.I. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3066-3070). In this paper, we have analyzed the structure of human gene for Gx alpha, which spans more than 60 kilobases. Comparison of the nucleotide sequence of the human chromosomal gene with that of the human retinal Gx alpha cDNA revealed that the gene is composed of three exons and two introns. The first exon contains about 170 base pairs of the 5'-noncoding sequence. The second exon contains the 5'-noncoding and the N-terminal coding sequences, and the third exon contains the C-terminal and the 3'-noncoding sequences. S1 mapping and primer extension analysis have identified the presence of multiple transcription initiation sites, upstream of which were found 12 SP1 binding sites and one CAAT sequence but no TATA sequence. Southern blot analysis indicated that a single copy of the Gx alpha gene is present per haploid human genome. RNA blot hybridization analysis revealed that Gx alpha mRNA is expressed mainly in brain.