Abstract: A novel, N-oligosaccharide-substituted-specific protein of approximately 91.5 kDa was purified from extracts of human articular cartilage and designated Cartilage Intermediate Layer Protein (CILP). The protein is a chondrocyte product which is deposited in the interterritorial matrix. The gene encoding CILP, as well as its mRNA, were isolated and characterized. A single 4.2 kb mRNA detected in human articular cartilage encodes a polypeptide of 1184 amino acids with a calculated molecular weight of 132.5 kDa. The protein has a putative signal peptide of 21 amino acids, and is a proform of two polypeptides. The amino-terminal half corresponds to CILP (molecular weight of 78.5 kDa, not including post-translational modifications) and the carboxy-terminal half corresponds to a protein homologous to a porcine nucleotide pyrophosphohydrolase, NTPPHase (molecular weight of 51.8 kDa, not including post-translational modifications).