In addition to its role in exocytosis, SNAP-25 is essential for axonal outgrowth. In order to identify SNARE proteins involved in neurite growth we have used SNAP-25 antibodies to affinity-purify protein complexes enriched in developing rat brain membrane extracts. We have identified a complex between SNAP-25 and syntaxin 13 predominantly present in brain at embryonic or early postnatal stages. We show that syntaxin 13 is developmentally regulated with a decrease in adult brain. In differentiated neuroendocrine PC12 cells as well as primary cortical neurons the protein is localized to a punctated and tubular staining in the perinuclear region and along processes with high levels in the central region of growth cones. Carboxy-terminally tagged syntaxin 13 was also detected on the plasma membrane by in vivo surface-labelling where it colocalized with SNAP-25. Syntaxin 13 has recently been shown to be implicated in early endosomal trafficking. In our study, colocalization with internalized transferrin in the cell body and along neurites confirmed endosomal location in both compartments. Finally, overexpression of full-length syntaxin 13 enhanced neurite outgrowth in NGF-stimulated PC12 cells, whilst it had no effect on regulated secretion. The data suggest that a syntaxin 13-dependent endocytic trafficking step plays a limiting role in membrane expansion during neuronal development.