Local cooling protects against TNF-alpha-induced injury by attenuating inflammation-associated microcirculatory dysfunction and leukocytic response. Mechanisms of protection, however, are not fully understood. We studied whether the metabolites of the HO and NOS pathway, exerting potent vasodilatory, antioxidant, and anti-apoptotic properties, are involved in tissue cryoprotection. In animals pretreated with L-NAME or SnPP-IX, cooling-associated abrogation of TNF-alpha-induced microcirculatory dysfunction was abolished. Combined L-NAME/SnPP-IX pretreatment did not cause greater blunting than seen when each mediator system was inhibited separately. In SnPP-IX- but not L-NAME-pretreated animals, transient hypothermia failed to reduce TNF-alpha-mediated leukocyte adherence. Vice versa, treatment of TNF-alpha-exposed animals with either the NO donor l-arginine or the HO-1 inductor hemin mimicked cooling-associated tissue protection except for failure of l-arginine to abrogate the inflammatory leukocyte response. The efficiency of cooling to inhibit TNF-alpha-induced apoptotic cell death was blunted in SnPP-IX-, L-NAME-, and SnPP-IX/L-NAME-pretreated animals. Coadministration of Trolox in SnPP-IX-treated animals partly attenuated leukocyte adherence and cell apoptosis, implying that the HO pathway metabolite biliverdin contributes to the salutary effects of cooling. Thus, our study provides evidence that metabolites of the HO and the NOS pathway mediate the cooling-associated protection of inflamed tissue. Biliverdin rather than CO and NO mediates the anti-inflammatory action, whereas a coordinated function of the gaseous monoxides prevents microcirculatory dysfunction and apoptotic cell death.