The present study tests the hypothesis that age-dependent increases in endothelial vasodilator capacity are due to maturational increases in endothelial NO synthesis and release. Intact 4-cm carotid artery segments taken from term fetal lambs and non-pregnant adult sheep were perfused using a closed system that enabled independent control of flow and inflow pressure, and facilitated complete recovery of all NO released. Fluid shear stress induced a graded release of NO (in nmol NO/min/cm² luminal surface area) that was significantly greater in adult (890±140) than fetal (300±40) carotids at corresponding values of shear stress (5.9±0.3 dynes/cm²), but was independent of inflow pressure in both age groups. These age-related differences in NO release were not attributable to corresponding differences in eNOS abundance, as eNOS protein levels (in ng eNOS / cm² luminal surface area) were similar in adult (14±2) and fetal (12±1) arteries. Adult (80±15) and fetal (89±32) levels of eNOS mRNA (in 106 copies / cm² luminal surface area) were also similar. However, when NO release was normalized relative to the associated mass of eNOS protein to estimate eNOS specific activity in situ, this value (in nmol NO/µg eNOS/min) was significantly greater in adult (177±44) than fetal (97±36) arteries when the endothelium was maximally activated by A23187. Similarly, the slope of the relation between fluid shear stress and estimated eNOS specific activity (in nmol NO/µg eNOS/min per dyne/cm²) was also significantly greater in adult (6.8±0.1) than fetal (2.9±0.1) arteries, suggesting that eNOS may be more sensitive to, or more efficiently coupled to, activating stimuli in adult compared to fetal arteries. We conclude that maturational increases in endothelial vasodilator capacity are attributable to age-dependent increases in NO release secondary to elevated eNOS specific activity and involve more efficient coupling between endothelial activation and enhancement of eNOS activity in adult compared to fetal arteries.