Chondrocytes are unique among non-transformed cells in that they are capable of anchorage-independent growth in soft agar. Fibroblast growth factor (FGF) is known as a potent colony-stimulating factor for chondrocytes. However, cartilage extracts contain a potent colony-stimulating activity which is not explicable only by contaminating FGF. We previously isolated the 25 kDa cartilage-specific glycoprotein chondromodulin-I (ChM-I) which stimulates the growth of chondrocytes. In the present study, we observed that ChM-I stimulates the colony formation of rabbit growth plate chondrocytes in agarose culture. ChM-I alone weakly stimulated the formation of chondrocyte colonies, but it markedly stimulated colony formation synergistically in the presence of an optimal dose of FGF-2. This effect was dependent on the dose of ChM-I. These results suggest that ChM-I participates in an autocrine signaling mechanism for the anchorage-independent growth of chondrocytes in vitro.