Syntaxins are transmembrane proteins that function in regulating transport vesicle docking and fusion with target membranes in neuronal and nonneuronal cells. Vesicle docking is thought to be regulated in part by the specific interactions of syntaxin with a vesicle-associated membrane protein termed synaptobrevin/VAMP. We have cloned a 1557-bp cDNA that encodes the human syntaxin 5 isoform, using a combination of PCR and colony-screening methods. The deduced 301 amino-acid sequence of human syntaxin 5 shares 96% identity with rat syntaxin 5. Like rat syntaxin 1A, human syntaxin 5 binds to synaptobrevin/VAMP in vitro. The identification of human syntaxin 5 as a synaptobrevin/VAMP-binding protein supports the hypothesis that syntaxin 5 regulates protein transport by binding to vesicle-associated membrane proteins.