We report here the detailed structure of a human beta-crystallin gene, designated Hu beta A3/A1, which was isolated and characterized using homologous mouse and bovine beta-crystallin cDNAs. Hu beta A3/A1 consists of six exons, spanning approximately 8 kilobases. The first two exons code for an N-terminal extension of 32 amino acid residues, while the other four encode the four similar structural motifs of the predicted polypeptide. Sequence homologies among the latter four exons and their intron-exon junctions support a model of gene evolution based on two successive exon duplications. Transcription of Hu beta A3/A1 in the eye lens initiates 24 base pairs downstream of a putative TATA box and just 7 nucleotides upstream of a potential initiation codon, generating a single mRNA of approximately 1 kilobase. Comparison of Hu beta A3/A1 with the homologous bovine cDNA and the translation products of the corresponding bovine gene suggests that translation of Hu beta A3/A1 commences at either of two potential initiation codons located in the first and second exons. Differential use of these two codons predicts two polypeptides differing by the presence or absence of 17 amino acid residues at their N-termini.