Collagen prolyl 4-hydroxylases (C-P4Hs) catalyze the formation of 4-hydroxyproline by the hydroxylation of -X-Pro-Gly-triplets. The vertebrate enzymes are alpha 2 beta 2 tetramers, the beta-subunit being identical to protein-disulfide isomerase (PDI). Two isoforms of the catalytic alpha-subunit, which combine with PDI to form [alpha(I)]2 beta 2 and [alpha(II)]2 beta 2 tetramers, have been known up to now. We report here on the cloning and characterization of a third vertebrate C-P4H alpha-subunit isoform, alpha(III). The processed human, rat and mouse alpha(III) polypeptides consist of 520-525 residues, all three having signal peptides of 19-22 additional residues. The sequence of the processed human alpha(III) polypeptide is 35-37% identical to those of human alpha(I) and alpha(II), the highest identity being found within the catalytically important C-terminal region and all five critical residues at the cosubstrate binding sites being conserved. The sequence within a region corresponding to the peptide-substrate binding domain is less conserved, but all five alpha helices constituting this domain can be predicted to be located in identical positions in alpha(I), alpha(II), and alpha(III) and to have essentially identical lengths. The alpha(III) mRNA is expressed in many human tissues, but at much lower levels than the alpha(I) and alpha(II) mRNAs. In contrast to alpha(I) and alpha(II), no evidence was found for alternative splicing of the alpha(III) transcripts. Coexpression of a recombinant human alpha(III) polypeptide with PDI in human embryonic kidney cells led to the formation of an active enzyme that hydroxylated collagen chains and a collagen-like peptide and appeared to be an [alpha(III)]2 beta 2 tetramer. The catalytic properties of the recombinant enzyme were very similar to those of the type I and II C-P4Hs, with the exception that its peptide binding properties were intermediate between those of the type I and type II enzymes.