Late development of endochondral ossification occurs at the boundary between the growth cartilage and bone marrow during the formation of long bones in Xenopus laevis. Since the Indian hedgehog (Ihh) is involved in endochondral ossification in mouse, we investigated the expression of Xenopus banded hedgehog (X-bhh), which is a homolog of mouse Ihh. RT-PCR analysis demonstrated that the X-bhh mRNA was detected from an early stage of limb formation to formation of femurs in mature frogs, and it was associated with the expression of Xenopus-ptc1 (X-ptc1), Xenopus-gli1 (X-gli1), Xenopus-type H collagen (X-col H), Xenopus-runx2 (Xrunx2), and Xenopus-osteocalcin (X-ocn) mRNAs. In situ hybridization revealed that chondrogenic cells observed at early limb development expressed X-bhh and X-gli1. At later stages of limb development, chondrocytes, located slightly away from the boundary between the cartilage and bone marrow, expressed the X-bhh, X-ptc1, and X-gli1 mRNAs; however, the mesenchymal cells at the boundary failed to express these mRNAs. The X-bhh, X-ptc1, and X-gli1 mRNAs as well as those of X-runx2 and X-ocn were expressed by the mesenchymal cells in the periosteal region at the tip of the cortical bone, indicating an intimate relationship between X-bhh expression and bone formation in this region. Considered collectively, the present study suggests that X-bhh evolutionally acquired the function to induce osteogenesis; however, the expression profile of X-bhh in epiphysis is closely related to the late development of endochondral ossification in X. laevis. (C) 2005 Elsevier Inc. All rights reserved.