Sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase 3 (SERCA3), an isoform of the intracellular Ca²⁺ pump that has been shown to mediate endothelium-dependent relaxation of vascular smooth muscle, is also expressed in tracheal epithelium. To determine its possible role in regulation of airway mechanical function, we compared tracheal contractility in gene-targeted mice deficient in SERCA3 (SERCA3⁻) with that in wild-type tracheae. Cumulative addition of ACh elicited concentration-dependent increases in isometric force (ED₅₀ = 2 μM, maximum force = 8 mN/mm²) that were identical in SERCA3⁻ and wild-type tracheae. After ACh stimulation, substance P (SP) elicited a transient relaxation (42.6 ± 3.2%, n = 28) in both tracheae. However, the rate of relaxation was significantly (P < 0.04,n = 9) more rapid in the wild-type [half-time (t _½) = 34.3 s] than in the SERCA3⁻(t _½ = 61.6 s) trachea. The SP relaxation was reduced by rubbing the trachea, indicative of epithelial cell involvement. This was verified using a perfused trachea preparation. SP in the outside medium had no effect, whereas SP in the perfusate bathing the epithelial side elicited a relaxation. Nitric oxide synthase inhibition (0.2 mMN ^ω-nitro-l-arginine) reduced the SP relaxation by 36.5 ± 12.5%, whereas the SP effect was abolished by eicosanoid inhibition (10 μM indomethacin). ATP also elicited an epithelium-dependent relaxation similar to SP but with a more rapid relaxation in the SERCA3⁻trachea than in the wild-type trachea. Our results indicate that SERCA3 gene ablation does not directly affect smooth muscle, which is consistent with the distribution of the isoform, but suggest that SERCA3 plays a role in epithelial cell modulation of airway smooth muscle function.