The Latent Transforming growth factor-beta Binding Proteins (LTBPs) are a family of extracellular matrix glycoproteins, some of which have dual functions as both regulators of Transforming Growth Factor-beta (TGF-beta) bioavailability and as structural extracellular matrix proteins. LTBP-3 is the smallest member of the LTBP family, and is known to bind TGF-beta strongly, but is one of the least well characterized in terms of structural and functional roles and patterns of expression. In order to gain insights into the functions of this molecule, several approaches were used; (a) to define LTBP-3 assembly and interactions with TGF-beta, and (b) to examine LTBP-3 expression and distribution within the growth plate of developing bone. A full-length LTBP-3 murine clone and LTBP-3 deletion constructs were utilised in in vitro translation studies, using a semi-permeabilised cell system. These studies have shown that the recombinant LTBP-3 undergoes N-glycosylation and readily forms disulphide bonded assemblies. Co-translation of LTBP-3 and TGF-beta indicates that LTBP-3 monomers can associate with the TGF-beta pro-peptide within the endoplasmic reticulum. During the course of this study, discrepancies were identified between the full-length LTBP-3 murine clone and the published sequence. Consequently, the correct sequence of murine LTBP-3 was established by extensive RT-PCR and sequencing studies. In situ hybridisation studies on fifteen-day-old mouse limb sections established the pattern of LTBP-3 expression in the growth plate. LTBP-3 was shown to be expressed by resting, proliferating, maturing and upper zone hypertrophic chondrocytes, but was not expressed by chondrocytes of the lower hypertrophic zone. These studies were confirmed by experiments using the differentiating chondrogenic cell line ATDC5, which is a useful in vitro culture model to examine the sequential stages of chondrogenic differentiation that occur within the epiphyseal growth plate. The data presented here, on this important TGF-beta binding molecule, have revealed the correct sequence of the murine LTBP-3 molecule, established its expression pattern in the growth plate and have shed new light on its ability to aggregate and interact with TGF-beta. This information contributes to our understanding of the biological roles of this key TGF-beta binding molecule.