By subtractive cDNA cloning we have identified a novel constituent of chicken cartilage termed matrilin-3. This constituent is encoded by a mRNA of 2.2 kbp whose expression is restricted to cartilaginous tissues. The predicted protein is composed of 452 amino acids with a molecular mass of 49 kDa. It contains a single von Willebrand factor A-like domain, four epidermal growth factor-like repeats and an alpha-helical region which may induce the formation of oligomers via a coiled-coil. The primary structure is similar to that of matrilin-1 which is also expressed in a cartilage-specific manner. This similarity suggests that the genes for the two proteins may have evolved from a common ancestor by gene duplication.