Previous studies have demonstrated that ribbon synapses in the retina do not contain the t-SNARE (target-soluble N-ethylmaleimide-sensitive factor attachment protein receptor) syntaxin 1A that is found in conventional synapses of the nervous system. In contrast, ribbon synapses of the retina contain the related isoform syntaxin 3. In addition to its localization in ribbon synapses, syntaxin 3 is also found in non-neuronal cells, where it has been implicated in the trafficking of transport vesicles to the apical plasma membrane of polarized cells. The syntaxin 3 gene codes for four different splice forms, syntaxin 3A, 3B, 3C and 3D. We demonstrate here using analysis of EST databases, RT-PCR, in situ hybridization and Northern blot analysis that cells in the mouse retina only express syntaxin 3B. In contrast non-neuronal tissues, such as kidney express only syntaxin 3A. The two major syntaxin isoforms (3A and 3B) have an identical N-terminal domain but differ in the C-terminal half of the SNARE domain and the C-terminal transmembrane domain. These two domains are thought to be directly involved in synaptic vesicle fusion. The interaction of syntaxin 1A and syntaxin 3B with other synaptic proteins was examined. We found that both proteins bind Munc18/N-sec1 with similar affinity. In contrast, syntaxin 3B had a much lower binding affinity for the t-SNARE SNAP-25 compared to that of syntaxin1A. Using an in vitro fusion assay we could demonstrate that vesicles containing syntaxin 3B and SNAP-25 could fuse with vesicles containing synaptobrevin2/VAMP2, demonstrating that syntaxin 3B can function as a t-SNARE.