A novel collagen IV chain, alpha 4(IV), has recently been identified in basement membranes. We describe part of the primary structure of the human alpha 4(IV) polypeptide for the first time, which has been determined by cloning and sequencing of cDNAs encoding 241 amino acid residues of the COL domain and 231 residues of the NC1 domain. We also characterized a genomic DNA fragment containing 4 exons coding for the entire NC1 domain. Among five known alpha chains of collagen IV, the alpha 4(IV) chain is distinct from the other four chains. However, it is more similar to the alpha 2(IV) chain than to the alpha 1(IV), alpha 3(IV) and alpha 5(IV) chains in terms of amino acid sequence homology, domain structure of polypeptides and exon/intron structure of the genes, suggesting the presence of two phylogenetically distinct subclasses of collagen IV alpha chains; one composed of alpha 2 and alpha 4 chains and the other of alpha 1, alpha 3 and alpha 5 chains.