In pancreatic beta-cells, the syntaxin 6 (Syn6) soluble N-ethylmaleimide-sensitive factor attachment protein receptor is distributed in the trans-Golgi network (TGN) (with spillover into immature secretory granules) and endosomes. A possible Syn6 requirement has been suggested in secretory granule biogenesis, but the role of Syn6 in live regulated secretory cells remains unexplored. We have created an ecdysone-inducible gene expression system in the INS-1 beta-cell line and find that induced expression of a membrane-anchorless, cytosolic Syn6 (called Syn6t), but not full-length Syn6, causes a prominent defect in endosomal delivery to lysosomes, and the TGN, in these cells. The defect occurs downstream of the endosomal branchpoint involved in transferrin recycling, and upstream of the steady-state distribution of mannose 6-phosphate receptors. By contrast, neither acquisition of stimulus competence nor the ultimate size of beta-granules is affected. Biosynthetic effects of dominant-interfering Syn6 seem limited to slowed intragranular processing to insulin (achieving normal levels within 2 h) and minor perturbation of sorting of newly synthesized lysosomal proenzymes. We conclude that expression of the Syn6t mutant slows a rate-limiting step in endosomal maturation but provides only modest and potentially indirect interference with regulated and constitutive secretory pathways, and in TGN sorting of lysosomal enzymes.