LTBP-2 is a matrix protein of unknown function since, unlike other LTBPs, it does not form covalent complexes with latent TGF-beta. We have previously shown that LTBP-2 has widespread association with fibrillin-containing microfibrils in developing aorta and other tissues. We have now shown that full-length human recombinant LTBP-2 specifically binds to the amino-terminal region of fibrillin-1, but not to fibrillin-2, in solid phase assays and overlay blotting. The binding was enhanced by the inclusion of 2 mM Ca2+ ions in the assay buffer and abolished by 5 mM EDTA indicating that the interaction was directly or indirectly Ca2+ ion dependent. The K(d) for the interaction was calculated from the specific binding curve as 9.4 nM. A recombinant carboxyl-terminal fragment of LTBP-2 was shown to a) bind the amino-terminal fragment of fibrillin-1 and b) block completely the binding of full length LTBP-2 to fibrillin-1. This result indicates that the major fibrillin-1 binding site resides close to the carboxyl-terminus of LTBP-2. Further competitive binding studies showed that an analogous carboxyl terminal fragment of LTBP-1 was able to block the binding of LTBP-2 to fibrillin-1 and that the C-terminal fragment of LTBP-2 could block the interaction of the LTBP-1 fragment with the fibrillin. Thus the binding site for LTBP-2 on fibrillin-1 appears to be the same or in close proximity to that for LTBP-1. Immunohistochemical analysis of developing human aorta showed distinctive but extensively overlapping distributions for LTBPs-1 and -2. Both LTBPs showed extensive co-localization with fibrillin-1 and elastic lamellae but LTBP-2 had extensive signal throughout the medial layer whereas LTBP-1 showed strong localization only in the outer medial layer. The finding indicates that there is a possibility for LTBP-2 to compete with LTBP-1 for binding to fibrillin-containing microfibrils throughout the aortic wall but particularly in the outer medial region where the LTBP-1 is predominantly located. Overall, the results support the concept that that LTBP-2 may be an indirect negative modulator for storage of the large latent TGF-beta complex on microfibrils in aorta and other fibrillin-rich tissues.