Type I procollagen COOH-terminal proteinase (C-proteinase) enhancer, a glycoprotein that binds to the COOH-terminal propeptide of type I procollagen and enhances procollagen C-proteinase activity, was purified from mouse fibroblast culture media. Partial amino acid sequences obtained from proteolytic fragments were found to have identity with the deduced amino acid sequence of a cDNA clone of unknown function, previously isolated from a mouse astrocyte library. Sequences of mouse enhancer cDNA, obtained in the present study, predict a approximately 50-kDa, 468-amino acid protein that differs from the 43-kDa, 402-amino acid protein predicted by the previously reported astrocyte-derived clone. Human cDNAs encode an enhancer of 449 amino acids. Previous biochemical studies have found the mouse enhancer as a 55-kDa form, which is readily processed to 36- and 34-kDa forms, retaining full C-proteinase enhancing activity and the ability to bind the COOH-terminal propeptide. Data presented here show the 36-kDa form to correspond to the amino-terminal portion of the 55-kDa protein. This is the most conserved region between mouse and human enhancers, comprising two domains with homology to domains found in a number of proteases and proteins with developmental functions. Such domains are thought to mediate interactions between proteins. Mouse enhancer RNA is shown to be at highest levels in collagen-rich tissues, especially tendon. The human enhancer gene, PCOLCE, is localized to 7q21.3-->q22, the same chromosomal region containing the type I collagen alpha 2 chain gene, COL1A2.