Tissues are often cold stored for physiological studies and for clinical transplantation. We report that cold storage induces a relaxation to reoxygenation after hypoxia (H/R) in de-endothelialized porcine coronary arteries. In fresh denuded arteries stimulated with U46619, H/R did not elicit relaxation. However, after overnight cold storage (4°C), H/R elicited a transient relaxation with peak relaxation of 56 ± 8% (n = 8), which was reproducible after 2 days of cold storage. The H/R relaxation was inhibited by methylene blue (10 µM) and LY83583 (10 µ M), O<sub>2</sub>-hemoglobin (1 µ M), or N<sup>G</sup>-methyl- L-arginine (0.2 m M), but neither N<sup>G</sup>-nitro- L-arginine (0.2 mM) nor cyclo-oxygenase inhibition was effective. Importantly, the H/R relaxation was attenuated by KCl (40 m M) or tetrabutylammonium chloride (5 m M), a non-selective inhibitor of K<sup>+</sup> channels. Interestingly, authentic nitric oxide (NO)- or S-nitroso-N-acetylpenicillamine (SNAP)-induced relaxations were enhanced by cold storage in U46619 (0.1 µ M) contractures. When tissues were contracted with KCl (40 m M), the enhancement in NO- or SNAP-induced relaxation by cold storage was markedly smaller than with U46619. Neither catalase (1,200 U/ml) nor 3-amino-triazole (50 m M), an inhibitor of catalase, affected the H/R relaxation. The duration of H/R relaxation also increased with the period of incubation at 37 ° C in the organ bath. This was blocked by inhibition of NO synthesis or guanylate cyclase. Moreover, inhibition of protein synthesis with actinomycin D (0.1 µ M) and cycloheximide (10 µ M), or dexamethasone (1 µ M), an inhibitor of NO synthase induction, blocked this increase in the duration of the H/R relaxation. The results suggest that in smooth muscle induction of NO pathway relaxation, which is in part mediated by K<sup>+</sup> channels and inducible NO synthase, may be of importance to the understanding of ischemia/reperfusion responses in cold-stored arteries.