An in vitro study of neural and myogenic control of human tracheal smooth muscle was undertaken. Over 80% of these had active tension and 13% had phasic contractile activity. Tonic and phasic activities were not reversed by indomethacin, 5,8,11,14-eicosatetraynoic acid, methysergide, mepyramine, atropine, or tetrodotoxin (TTX) but were blocked by the calcium antagonist verapamil. In some quiescent strips, tonic and/or phasic activity was induced by exposure to potassium-conductance blockers such as 4-aminopyridine (4-AP) and tetraethylammonium chloride (TEA). Electrical (field) stimulation resulted in frequency-dependent biphasic responses: an initial atropine-sensitive cholinergic contraction followed by a nonadrenergic relaxation. This biphasic response to low stimulus parameters (less than 0.5 ms, less than 15 Hz) was blocked by TTX and scorpion venom and enhanced by 4-AP and TEA, consistent with a neural mechanism. Relaxation responses to longer pulse durations (0.5–1 ms) were not blocked by TTX despite abolition of contraction nor were they enhanced by 4-AP and TEA, suggesting a nonneural mechanism. ATP, adenosine, arachidonate metabolites, histamine, 5-hydroxytryptamine, neurotensin, or vasoactive intestinal polypeptide were ruled out as possible nonadrenergic mediators. The nature and physiological significance of the nonneural inhibitory response remains unknown.