The mechanisms through which p38 mitogen-activated protein kinase (p38 MAPK) is involved in smooth muscle contraction remain largely unresolved. We examined the role of p38 MAPK in prostaglandin F₂ (PGF₂)-induced vasoconstriction and in hypoxic pulmonary vasoconstriction (HPV) of rat small intrapulmonary arteries (IPA). The p38 MAPK inhibitors SB203580 and SB202190 strongly inhibited PGF₂-induced vasoconstriction, with IC₅₀s of 1.6 and 1.2 µM, whereas the inactive analogue SB202474 was ~30-fold less potent. Both transient and sustained phases of HPV were suppressed by SB203580, but not by SB202474 (both 2 µM). Western blot analysis revealed that PGF₂ (20 µM) increased phosphorylation of p38 MAPK and of heat shock protein 27 (HSP27), and this was abolished by SB203580 but not by SB202474 (both 2 µM). Endothelial denudation or blockade of eNOS with L-NAME significantly suppressed the relaxation of PGF₂-constricted IPA by SB203580, but not by SB202474. Similarly, the inhibition of HPV by SB203580 was prevented by prior treatment with L-NAME. 2 µM SB203580, but not SB202474 enhanced relaxation induced by the NO donor S-nitroso-N-acetylpenicillamine (SNAP) in endothelium-denuded IPA constricted with PGF₂. In -toxin permeabilized IPA, SB203580-induced relaxation occurred in the presence but not the absence of the NO donor sodium nitroprusside (SNP); SB202474 was without effect even in the presence of SNP. In intact IPA, neither PGF₂ nor SNAP-mediated changes in intracellular [Ca²⁺] were affected by SB203580. We conclude that p38 MAPK contributes to PGF₂- and hypoxia-induced constriction of rat IPA primarily by antagonizing the underlying Ca²⁺-desensitizing actions of NO.