It has long been established that LTBP-2 is associated with elastic fibres during the period of elastogenesis. Its function remains largely unknown although it does bind fibrillin-1, fibulin-5 and heparin/heparan sulphate in vitro but has no affinity for tropoelastin. The aim of this current study was to determine if LTBP-2 enhanced or inhibited tropoelastin interactions with fibulin-5 and heparin. Firstly, using solid phase binding assays we showed that LTBP-2 bound fibulin-2 with an affinity similar to that of the tropoelastin - fibulin-5 interaction. (Kds = 47 nM and 39 nM respectively). Then using a competitive binding assay we showed that LTBP-2 inhibited the tropoelastin-fibulin-5 interaction in a dose dependent manner with almost complete inhibition obtained with 5-fold molar excess of LTBP-2. Interestingly, a fragment of LTBP-2 containing the fibulin-5 binding sequence only partially inhibited the tropoelasin-fibulin-5 interaction suggesting that LTBP-2 was directly blocking only the C-terminal tropoelastin binding site on fibulin-5 and indirectly blocking tropoelastin binding to the N-terminal region. In a similar assay, LTBP-2 was shown to partially inhibit the binding of heparin to tropoelastin with 50% inhibition achieved with 10 fold molar excess of LTBP-2. Confocal microscopy of fibroblast matrix showed strong co-distribution of LTBP-2 with fibulin-5 and fibrillin-1 and partial codistribution with heparan sulphate proteoglycans, perlecan and syndecan-4. Overall the results suggest that LTBP-2 may be involved in displacing elastin microassemblies from complexes with fibulin-5 and/or cell surface heparan sulphate during elastic fibre deposition.