Asporin is an extracellular matrix (ECM) protein that regulates cartilage matrix gene expression and cartilage formation by modulating the transforming growth factor-beta (TGF-beta) signaling pathway. Our previous studies have indicated that asporin binds to TGF-beta1 directly and inhibits TGF-beta1-mediated expression of cartilage matrix genes. However, it is still unknown how asporin interacts with TGF-beta1 and influences its activity. Using competition assays, we determined that amino acids 159-205 of asporin mediate its interaction with TGF-beta1 and effectively repress TGF-beta1-induced cartilage matrix gene expression. Asporin also has a binding ability to type II collagen in vitro, but its binding pattern is different from that of TGF-beta1. In contrast with previous in vivo findings, asporin did not affect the interaction between TGF-beta1 and the TGF-beta type II receptor (TbetaRII) by itself or in the presence of type II collagen in vitro. However, in the presence of heparin/heparan sulfate, asporin inhibits the interaction between TGF-beta and TbetaRII in vitro. These findings suggest that asporin is one of the important cartilage matrix proteins that binds to the ECM and TGF-beta1 and thereby modulates interactions between TGF-beta and its signaling receptors.