betaig-h3 is a TGF-beta-induced extracellular matrix protein which is expressed in many tissues including bones and cartilages. In previous reports, we showed that betaig-h3 mediates cell adhesion and migration and, especially in bones, negatively regulates the mineralization in the end stage of endochondral ossification. Here, to elucidate the expression pattern and role of betaig-h3 in chondrocyte differentiation, ATDC5 chondrocytes and embryonic and postnatal mice were used for in vitro differentiation studies and in vivo studies, respectively. betaig-h3 was strongly induced by the treatment of TGF-beta1 and the expression level of betaig-h3 mRNA and protein were highly expressed in the early stages of differentiation but decreased in the late stages in ATDC5. Furthermore, the patterns of TGF-beta1, -beta2, and -beta3 mRNA expression were concurrent with betaig-h3 in ATDC5. betaig-h3 was deeply stained in perichondrium (PC), periosteum (PO), and prehypertrophic chondrocytes (PH) through the entire period of endochondral ossification in mice. betaig-h3 was mainly expressed in PC and PH at embryonic days and obviously in PH in postnatal days. These results suggest that betaig-h3 may play a critical role as a regulator of chondrogenic differentiation in endochondral ossification.