We have isolated a cDNA clone for human matrilin-3 from a cartilage-specific cDNA library. The polypeptide predicted from the nucleotide sequence of this clone shared 83% identity with matrilin-3 from mouse and 61% with that from chicken. It was composed of 486 amino acid residues that were arranged in seven domains: a signal peptide, a von Willebrand factor A domain, four EGF repeats, and an α-helical region. The gene for human matrilin-3 (MATN3) was assigned to chromosome region 2p24–p23. The corresponding mRNA of 2.8 kb was expressed in every type of cartilage investigated thus far. It was also producedin vitroby primary chondrocytes isolated from articular cartilage. However, dedifferentiated chondrocytes of the third passage did not express it at all. Matrilin-3 might therefore serve as a marker for the differentiation state of chondrocytes.