Two complementary DNA clones pRL gamma-2 and pRL gamma-3 of different rat lens gamma-crystallin messenger RNAs have been used to identify gamma-crystallin gene sequences in rat genomic DNA. Subsequently, the DNA present in the 18,000 to 20,000 bases region of the EcoRI digest, giving rise to a strong doublet hybridization signal, was cloned in lambda phage Charon-4A. One of the clones, lambda RCH gamma-3, carrying an insert of 17,500 bases has been characterized in detail. From analysis at the restriction enzyme level with 5'-, "middle" and 3'-specific subprobes of pRL gamma-3 it could be deduced that lambda RCH gamma-3 contains only one gamma-crystallin gene. The coding sequences of this gene are interrupted by intronic DNA. The primary structure of this gene and its flanking regions have been established by sequencing the relevant regions of a subclone of lambda RCH gamma-3, designated pRCH gamma-3 . 1. The sequence data show that the gamma-crystallin gene extends over 2700 bases of rat genomic DNA. The gene is split by two introns, one of 87 base-pairs after the third translation codon and a large one of 1880 base-pairs after codon 84. The mosaic structure of the gene is strictly co-linear with the structure of the gamma-crystallin polypeptide in that the large intron is positioned in a region which specifies the so-called "connecting peptide" and which links the two highly symmetrical and homologous protein domains. Although expected from the cDNA and protein sequence no introns were observed between the coding regions in the DNA specifying the two homologous folding motifs present in each protein domain. The relevance of this phenomenon in terms of the evolution of the mature gamma-crystallin gene is discussed.