This work reports the first direct observations of binding and complex formation between transforming growth factor beta 1 (TGF-beta 1) and cartilage oligomeric matrix protein (COMP) using high-resolution atomic force microscopy (AFM). Each COMP molecule consists of pentamers whose five identical monomeric units bundle at N-termini. From this central point, the five monomers' flexible arms extend outward with C-terminal domains at the distal ends, forming a bouquet-like structure. In commonly used buffer solutions, TGF-beta 1 molecules typically form homodimers (majority), double dimers (minority), and aggregates (trace amount). Mixing TGF-beta 1 and COMP leads to rapid binding and complex formation. The TGF-beta 1/COMP complexes contain one to three COMP and multiple TGF-beta 1 molecules. For complexes with one COMP, the structure is more compact and less flexible than that of COMP alone. For complexes with two or more COMP molecules, the conformation varies to a large degree from one complex to another. This is attributed to the presence of double dimers or aggregates of TGF-beta 1 molecules, whose size and multiple binding sites enable binding to more than one COMP. The number and location of individual TGF-beta 1 dimers are also clearly visible in all complexes. This molecular-level information provides a new insight into the mechanism of chondrogenesis enhancement by TGF-beta 1/COMP complexes, i.e., simultaneous and multivalent presentation of growth factors. These presentations help explain the high efficacy in sustained activation of the signaling pathway to augment chondrogenesis.