PRG4 (proteoglycan 4) has been identified as megakaryocyte stimulating factor and articular superficial zone protein. PRG4 has characteristic motifs including somatomedin B and hemopexin domains, a chondroitin sulfate-attachment site and mucin-like repeats. During a screen of genes implicated in ectopic ossification, we found a novel mouse gene highly homologous to human and bovine PRG4 genes. Here, we report isolation, characterization and mapping of the gene, Prg4 together with characterization of its human orthologue. Prg4 cDNA was 3,320 bp long, encoding a 1,054 amino-acid protein. Human and mouse PRG4 genes each consisting of 12 exons spanned 18 and 16 kb, respectively. Characteristic motifs were conserved across species; however, the mucin-like repeat regions were highly diverse in length between species with a tendency that larger animals had longer repeats. Expression of human and mouse PRG4 genes was similar and found not only in cartilage, but also in liver, heart, lung, and bone. Expression of the mouse gene increased with progression of ectopic ossification. Multiple tissue-specific splicing variants lacking some of the motifs were found in both human and mouse. Although a specific role in the articular joint has previously been reported, the presence of multi-functional motifs as well as unique expression and alternative splicing patterns suggest that PRG4 functions in several distinctive biological process including regulation of ossification.