Members of the MAP kinase family of enzymes, p42/44 and p38, have been implicated in both regulation of contractile function and growth responses in vascular smooth muscle. We determined whether such kinases are activated during the arteriolar myogenic response following increases in intraluminal pressure. Particular emphasis was placed on temporal aspects of activation to determine whether such phosphorylation events parallel the known time course for myogenic contraction. Studies used single cannulated arterioles isolated from cremaster muscle of rats with some vessels loaded with the fluorescent Ca²⁺ sensitive dye, fura 2 (2 µM). The p42/44 inhibitor, PD 98059 (50 µM), caused vasodilation but did not prevent pressure-induced myogenic constriction. The vasodilator response was accompanied by decreased smooth muscle intracellular Ca²⁺ . Western blotting revealed a significant increase in the level of phosphorylation of p42/44, 15 minutes after the application of a 30 to 100 mmHg pressure step. Phosphorylation of p42/44 was a late event that was temporally dissociated from contraction which was complete within 1 to 5 minutes. EGF (80nM) caused marked phosphorylation of p42/44 but only acted as a weak vasoconstrictor. The p38 inhibitor, SB203580 (10µM), did not alter baseline diameter nor did it prevent myogenic vasoconstriction. Consistent with these observations SB203580 did not cause a measurable change in intracellular Ca²⁺. The results demonstrate activation of the p42/44 class of MAPK resulting from increased transmural pressure. Such activation is, however, dissociated from the acute pressure-induced vasoconstrictor response in terms of time course and may represent activation of compensatory, but parallel, pathways including those related to growth and remodeling.