The complete amino acid sequence of the triple-helical domain of human collagen VI was deduced from sequences of appropriate cDNA clones and confirmed to about 50% by Edman degradation of tryptic peptides. This domain consists of three different peptide segments containing some 335-336 amino acid residues originating from central portions of the alpha 1 (VI), alpha 2(VI), and alpha 3(VI) chains, respectively. Sequence identity in the X/Y positions of the Gly-X-Y repeats is rather low (10-15%) between the chains. Peculiar features of these sequences include 3 cysteine residues about 50 (alpha 3(VI)) and 89 (alpha 1(VI), alpha 2(VI)) residues away from the N-terminus and several Gly-X-Y interruptions clustered in the C-terminal two-thirds of the triple helix. These structures are presumably required for cross-linking collagen VI oligomers and for super-coiling of triple helices in the dimers. Other features include 11 Arg-Gly-Asp sequences, some of which are likely to be used as cell-binding sites, and four Asn-X-Thr sequences, allowing N-linked glycosylation along the triple helix. Junctional areas close to the helix contain short, cysteine-rich segments which may seal the triple-helical domain through disulfide bond formation, endowing it with high stability. These features, together with a low sequence homology to fiber-forming and basement-membrane collagens, document the unique character of collagen VI, whose triple helix is specifically adjusted for forming microfibrils in tissues.