We studied the effect of vasoactive intestinal peptide (VIP) on the output of ³⁵S-labeled macromolecules from ferret tracheal explants either placed in beakers or suspended in modified Ussing chambers. In Ussing chamber experiments, the radiolabel precursor, sodium [³⁵S]sulfate, and all drugs were placed on the submucosal side of the tissue. Washings were collected at 30-min intervals from the luminal side and were dialyzed to remove unbound ³⁵S, leaving radiolabeled macromolecules. Vasoactive intestinal peptide at 3 × 10⁻⁷ M stimulated bound ³⁵S output by a mean of +252.6% (n = 14). The VIP response was dose-dependent with a near maximal response and a half maximal response at approximately 10⁻⁶ M and 10⁻⁸, M, respectively. The VIP effect was not inhibited by a mixture of tetrodotoxin, atropine, l-propranolol, and phentolamine. Vasoactive intestinal peptide had no effect on the electrical properties of the tissues. We conclude that VIP stimulates output of sulfated-macromolecules from ferret tracheal submucosal glands without stimulating ion transport. Our studies also suggest that VIP acts on submucosal glands via specific VIP receptors. Vasoactive intestinal peptide has been shown to increase intracellular levels of cyclic AMP, and we suggest that this may be the mechanism for its effect on the output of macromolecules. This mechanism may be important in the neural regulation of submucosal gland secretion.