The integrin β7 subunit associates with two alternative α subunits termed αHML-1 and α4 to give the lymphocyte activation and homing receptors HML-1 and LPAM-1. Overlapping genomic clones that encoded the human (β7 subunit gene were isolated from cosmid and phage λ libraries. The coding portion of the gene spanned ∼10 kb and was composed of 14 exons. Exon 1 (123 bp) encoded 5' untranslated sequences; exon 2 (204 bp) encoded the initiation codon, signal peptide, and 50 amino acid residues of the N-terminus of the mature protein; 7 exons (exons 3-9), ranging in size from 90 bp to 242 bp, encoded most of the extracellular domain proximal to the four cysteine-rich repeats; the region corresponding to the β3 arginine-glycine-aspartic acid (RGD)-binding domain was divided amongst exons 4 and 5; the four cysteine-rich repeats were encoded by 3 exons (exons 10-12) with intron insertion into the first and third repeats; exon 13 (209 bp) provided a spacer between the cysteine-rich domains and the transmembrane domain; exon 14 (161 bp) encoded the transmembrane domain and exactly half of the cytoplasmic domain; the remainder of the cytoplasmic domain and most of the 3' untranslated region was contained in the largest 313 bp exon 15. Comparison of integrin β subunit genes revealed that the gene organization of (β7 was almost identical to that of β2, but had diverged from that of β3. Amplification of integrin DNAs directly from genomic DNA, using PCR primers based on β subunit consensus sequences corresponding to the β3 RGD-binding domain, yielded partial gene sequences for the β3, β5, and β6 subunits only. Inspection of the amplified sequences revealed that, as for β3, the regions in β5, and β6, corresponding to the β3, RGD-binding domain lacked the intron present in β7, β1, and β2, which divides this region in β2, into two subdomains that contribute to subunit assembly. This study provides genetic evidence for at least two major branches to the integrin β subunit evolutionary tree, with β7, β2, and probably β1, in one branch, and the cytoadesin β3, and probably also β5, and β6, in the other.