The present study explores the hypothesis that arterial smooth muscle cells are organized into layers with similar phenotypic characteristics that vary with the relative position between the lumen and the adventitia due to transmural gradients in vasotrophic factors. A corollary hypothesis is that vascular endothelial growth factor (VEGF) is a factor that helps establish transmural variations in smooth muscle phenotype. Organ culture of endothelium-denuded ovine carotid arteries with 3 ng/ml VEGF-A(165) for 24 h differentially and significantly influenced potassium-induced (55% increase) and stretch-induced (36% decrease) stress-strain relations in adult (n = 18) but not term fetal (n = 21) arteries, suggesting that smooth muscle reactivity to VEGF is acquired during postnatal maturation. Because inclusion of fetal bovine serum significantly inhibited all contractile effects of VEGF (adult: n = 11; fetus: n = 11), it was excluded in all cultures. When assessed in relation to the distance between the lumen and the adventitia in immunohistochemically stained coronal artery sections, expression of smooth muscle α-actin (SMαA), myosin light chain kinase (MLCK), and 20-kDa regulatory myosin light chain exhibited distinct protein-dependent and age-dependent gradients across the artery wall. VEGF depressed regional SMαA abundance up to 15% in adult (n = 6) but not in fetal (n = 6) arteries, increased regional MLCK abundance up to 140% in fetal (n = 8) but not in adult (n = 10) arteries, and increased regional MLC(20) abundance up to 28% in fetal arteries (n = 7) but decreased it by 17% in adult arteries (n = 9). Measurements of mRNA levels verified that VEGF receptor transcripts for both Flt-1 and kinase insert domain receptor (KDR) were expressed in both fetal and adult arteries. Overall, the present data support the unique hypothesis that smooth muscle cells are organized into lamina of similar phenotype with characteristics that depend on the relative position between the lumen and the adventitia and involve the direct effects of growth factors such as VEGF, which acts independently of the vascular endothelium in an age-dependent manner.