The integrin beta 7 subunit associates with two alternative alpha subunits termed alpha HML-1 and alpha 4 to give the lymphocyte activation and homing receptors HML-1 and LPAM-1. Overlapping genomic clones that encoded the human beta 7 subunit gene were isolated from cosmid and phage lambda libraries. The coding portion of the gene spanned approximately 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 beta 3 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 beta subunit genes revealed that the gene organization of beta 7 was almost identical to that of beta 2, but had diverged from that of beta 3. Amplification of integrin DNAs directly from genomic DNA, using PCR primers based on beta subunit consensus sequences corresponding to the beta 3 RGD-binding domain, yielded partial gene sequences for the beta 3, beta 5, and beta 6 subunits only. Inspection of the amplified sequences revealed that, as for beta 3, the regions in beta 5 and beta 6 corresponding to the beta 3 RGD-binding domain lacked the intron present in beta 7, beta 1, and beta 2, which divides this region in beta 2 into two subdomains that contribute to subunit assembly. This study provides genetic evidence for at least two major branches to the integrin beta subunit evolutionary tree, with beta 7, beta 2, and probably beta 1 in one branch, and the cytoadhesin beta 3 and probably also beta 5 and beta 6 in the other.