We have characterized cDNA clones that encode a newly discovered collagenous polypeptide. A 4-kilobase (kb) cDNA clone was initially isolated by screening a human fibroblast cDNA library with a probe encoding the collagenous domain of the human alpha 3(VI) collagen. Subsequent screening of another fibroblast cDNA library yielded overlapping clones having a total length of 5.4 kb, which contained an open reading frame of 1603 amino acids including a 21-amino acid signal peptide. The predicted polypeptide consists of 10 collagenous domains 15-422 amino acids long, which were interspersed with 11 noncollagenous (NC) domains. Except for a large N-terminal NC11 domain of 312 residues, most of the NC domains were short (11-39 residues) and cysteine-rich. The overall structural arrangement differed significantly from other known collagen chains. Further analysis indicated that the deduced polypeptide exhibited several structural features characteristically seen in members of the fibril-associated collagen, types IX, XII, and XIV. In addition, the cysteine-rich motifs in the NC domains resembled those found in the cuticle collagen of Caenorhabditis elegans. Northern blot analyses showed hybridization of the cDNA to a 5.5-kb mRNA in human fibroblasts and keratinocytes. The gene was localized by in situ hybridization to band p34-35 of human chromosome 1. The data clearly support the conclusion that the cDNA encodes a collagen chain that has not been previously described. We suggest that the cDNA clones encode the alpha 1 chain of type XVI collagen.