5-Hydroxytryptamine (5-HT) activates the extracellular signal-regulated kinase (Erk) mitogen-activated protein kinases (MAPKs) in the vasculature, resulting in contraction. The mechanisms by which this occurs are unclear. G protein-coupled receptors can activate Erk MAPK pathways through a variety of mechanisms, including stimulation of Src, phosphoinositide-3 kinase (PI-3-K), protein kinase C (PKC), or the epidermal growth factor (EGF) receptor tyrosine kinase. We hypothesize that 5-HT uses one or more of these pathways. In isolated strips of rat aorta, the MAPK/Erk kinase inhibitor U0126 (50 microM), Src inhibitor PP1 (0.5 microM), PKC inhibitors calphostin C (1 microM) and chelerythrine (10 microM), and the PI-3-K inhibitor LY294002 (1-20 microM) reduced 5-HT-induced contraction. The EGF receptor tyrosine kinase inhibitor AG1478 (0.25-1 microM) was without effect. Thus, 5-HT activates PKC, Src, and possibly PI-3-K to result in contraction. In rat aortic myocytes, 5-HT (1 microM) activated Erk MAPK proteins 2- to 3-fold over basal values; activation was reduced by U0126, PP1, and LY294002 and unaffected by calphostin C or chelerythrine, wortmannin, or AG1478. The lack of effect of EGF receptor tyrosine kinase and PI-3-K inhibitors was confirmed in that the EGF receptor immunoprecipitated from 5-HT-exposed cells did not display an increase in autophosphorylation, nor did 5-HT significantly increase activation of Akt/protein kinase B, a downstream substrate for PI-3-K. These data suggest that the rat aortic 5-HT(2A) receptor uses Src but not PKC, PI-3-K, or the EGF receptor tyrosine kinase in stimulating Erk MAPK activation.