To distinguish the origin of bone-forming cells in the osteosarcoma (OST) tumor inoculated into nude mice, we have developed a novel in situ hybridization technique. The system used digoxygenin (DIG) labeled DNA probes that encoded human specific repetitive gene, Alu, and mouse specific repetitive gene, mouse L1 (m-L1). The chondrogenic and osteogenic cells in the tumor had strongly positive signals for m-L1 probe without any signals for Alu probe. The expression of bone matrix proteins was also examined by in situ hybridization. The bone-forming cells were positive for mRNAs of mouse osteonectin, osteopontin, and osteocalcin relating to calcification during bone formation, while these were negative for human mRNAs of these bone matrix proteins. The OST cells in the tumor expressed the human bone morphogenetic proteins (BMPs) mRNAs by RT-PCR. These data indicated that the mouse cells, not the human sarcoma cells, are responsible for cartilage and bone formation in the OST tumor inoculated into nude mice, and we speculated that BMPs, at least in part, could play an important role in this ossification.