Major intrinsic protein (MIP, also called MP26) is the predominant fiber cell membrane protein of the ocular lens. MIP has been suggested to play a role in cell-cell communication in the lens. Its expression is tissue-specific and developmentally regulated. We have isolated and characterized the human gene encoding MIP and report here its genomic structure and entire nucleotide sequence. The gene is 3.6 kb, contains four exons separated by introns ranging in size from 0.4 to 1.6 kb, and is present in single copy per haploid human genome. Primer extension of human lens RNA indicates that transcription of the gene initiates from a single site 26 nt downstream from the TATA box. Three complete Alu repetitive elements are found in tandem in the 5'-flanking region of the gene, and a single complete Alu sequence is present in the third intron. The interspecies comparisons of the MIP gene coding sequence and homologies to other members of a putative transmembrane channel protein superfamily are also discussed.