Synaptophysin (SYP) is a major protein of neurotransmitter-containing vesicles spanning the membrane four times and contributing to various aspects of the synaptic vesicle cycle. The split-ubiquitin yeast two-hybrid system was used to characterize molecular interactions of membrane-bound, full-length murine SYP. In this way, the known homophilic SYP-SYP association could be confirmed and heterophilic binding of SYP to other tetraspan vesicle membrane proteins of the secretory carrier-associated membrane- and synaptogyrin-type could be detected for the first time. SYP-binding was also observed for the vSNARE synaptobrevin2 and various membrane and membrane-associated proteins. Double labeling immunofluorescence microscopy of murine retina, co-immunoprecipitation experiments and fluorescence energy resonance transfer (FRET) analyses between fluorescent protein-tagged polypeptides were carried out to validate and further characterize the association of SYP with the tetraspan vesicle membrane proteins secretory carrier-associated membrane protein 1 and synaptogyrin3, with synaptobrevin2, and the newly identified binding partners phospholipase D4, stathmin-like3, Rho family GTPase2 and ADP-ribosylation factor interacting protein2. It was observed that the carboxyterminus of SYP is dispensable for association with integral membrane proteins while it is needed for binding to membrane-associated polypeptides. The latter appears to be regulated by phosphorylation, since src homology 2-domains were shown to attach to the multiple carboxyterminal phosphotyrosine residues of SYP. In conclusion, the association of SYP with different tetraspan vesicle membrane proteins suggests shared functions and the multiple other interactions identify SYP as part of a membrane platform acting as a facilitator of various steps of the synaptic vesicle cycle.