Syntaxin 1 and synaptobrevin play an essential role in synaptic vesicle exocytosis. Two isoforms for each of these proteins, syntaxin 1A and 1B and synaptobrevin 1 and 2, have been found in nerve endings. Previous morphological studies have revealed a characteristic co-localization of syntaxin 1 and synaptobrevin isoforms in nervous and endocrine systems; however, the physiological significance of differential distribution is not known. In the present study an in vitro assay has been used to study a possible isoform specific interaction between syntaxin and synaptobrevin isoforms. The results show that although both syntaxin 1A and 1B may interact with synaptobrevin 1 and 2, this interaction is not uniform, showing different affinity patterns depending on the syntaxin 1/synaptobrevin isoform combination. The addition of SNAP-25 increased the binding capacity of syntaxin and synaptobrevin isoforms without affecting specific interactions.