Crk-associated substrate (CAS), a 130-kDa adaptor protein, was discovered as a tyrosine kinase substrate of Src that was important to cellular motility and actin filament formation. As the tyrosine kinase Src is utilized by the 5-hydroxytryptamine (5-HT)(2A) receptor in arterial contraction, we tested the hypothesis that CAS was integral to 5-HT(2A) receptor-mediated vasoconstriction. Rat thoracic aorta was used as a model of the arterial 5-HT(2A) receptor. Western and immunohistochemistry analyses validated the presence of CAS in the aorta, and tissue bath experiments demonstrated reduction of contraction to 5-HT (13.5 +/- 5% control maximum) and the 5-HT(2) receptor agonist alpha-methyl-5-HT (6 +/- 2% maximum) by latrunculin B (10(-6) mol/l), an actin disruptor. In aorta contracted with 5-HT (10(-5) mol/l), tyrosine phosphorylation (Tyr410) of CAS was significantly increased (approximately 225%), and both contraction and CAS phosphorylation were reduced by the 5-HT(2A/2C) receptor antagonist ketanserin (3 x 10(-8) mol/l). Src is one candidate for 5-HT-stimulated CAS tyrosyl-phosphorylation as 5-HT promoted interaction of Src and CAS in coimmunoprecipitation experiments, and the Src tyrosine kinase inhibitor PP1 (10(-5) mol/l) abolished 5-HT-induced tyrosyl-phosphorylation of CAS and reduced 5-HT- and alpha-methyl-5-HT-induced contraction. Antisense oligodeoxynucleotides delivered to the aorta reduced CAS expression (33% control) and arterial contraction to alpha-methyl-5-HT (45% of control), independent of changes in myosin light chain phosphorylation. These data are the first to implicate CAS in the signal transduction of 5-HT.