Male Wistar rats were humanely killed and IPA (internal diameter 200-500μm) were used in all experiments. Student’s t test was used for all statistical comparisons, and data are presented as mean ± S.E.M. IPA constricted with 20 μM PGF_2α were relaxed by SB203580 with an apparent IC₅₀ of 1.6 ± 0.4 μM (n = 12) and a near-maximal effect at 30 μM. The inactive analogue SB202474 was approximately 30-fold less potent (only 2.8 ± 1.4% relaxation at 1 μM and only 39 ± 6% relaxation at 20 μM, n = 10). SB203580 also inhibited the second phase contractile response to hypoxia (∼50% inhibition, n = 8, P < 0.05), while SB203474 had no effect. PGF_2α significantly increased levels of the phosphorylated forms of p38 MAPK (168 ± 20% of control, n = 16, P < 0.01) and of its downstream effector, heat shock protein 27 (HSP27) (278 ± 77% of control, n = 21, P < 0.05) in IPA. Both increases were reversed by co-incubation with 2 μM SB203580 (77 ± 7% of control for p38 MAPK, n = 5; 71 ± 16% of control for HSP27, n = 8), but not by 2μM SB202474 (194 ± 53% of control for p38 MAPK, n = 7; 276 ± 66% of control for HSP27, n = 8). The sensitivity of the PGF_2α contraction to SB203580 was greatly reduced by endothelial denudation (IC₅₀ 16 ± 2.3 μM, n = 10, P < 0.001 vs. control). The response to SB202474, however, was not affected by endothelial denudation. The sensitivity of PGF_2α contraction to SB203580 was also inhibited by 1 mM L-NAME (IC₅₀ = 12 ± 3 μM, n = 13, P < 0.01 vs. control), indicating the involvement of nitric oxide in the relaxation response. Similarly, L-NAME prevented the inhibition of the second phase of HPV by SB203580. As an indication of NO bioavailability, we measured cGMP production in HUVECs. Levels of cGMP were increased 2- to 3-fold by both SB203580 and SB20474 (2 μM, n = 8), although this was not observed in the presence of PGF_2α, which itself also increased cGMP levels. This suggests that p38 MAPK is not specifically involved in either NO production or regulating guanylate cyclase activity. As an indicator of smooth muscle sensitivity to NO, the influence of p38 MAPK inhibition on relaxation of PGF_2α-contracted endothelium-denuded arteries to the NO donor SNAP was examined. 2 μM SB203580, but not SB202474, enhanced the relaxation response to SNAP (IC₅₀: control, 0.28 ± 0.11 μM, n = 8, +SB203580, 0.12 ± 0.03 ± 0.03 μM, P < 0.05; maximum relaxation: control, 56 ± 8.4%, +SB203580, 82.8 ± 6%, P < 0.05). These results suggest that p38 MAPK activity contributes to PGF_2α-induced and hypoxia-induced contraction of IPA partly via inhibition of the relaxing actions of nitric oxide.