Postnatal decreases in vascular reactivity involve decreases in the thick filament component of myofilament calcium sensitivity, which is measured as the relationship between cytosolic calcium concentration and myosin light chain (MLC20) phosphorylation. The present study tests the hypothesis that downregulation of thick filament reactivity is due to downregulation of myosin light chain kinase (MLCK) activity, in adult compared to fetal arteries. Total MLCK activity, calculated as % MLC20 phosphorylated/sec in intact arteries during optimal inhibition of myosin light chain phosphatase activity, was significantly less in adult (6.56±0.29%) than in fetal (7.39±0.53%) preparations. In-situ MLC20 concentrations (µM) in adult (198±28) and fetal (236±44) arteries did not differ significantly. In-situ MLCK concentrations (µM), however, were significantly greater in adult (8.21±0.59) than fetal (1.83±0.13) arteries. In-situ MLCK activities in ng MLC20 phosphorylated/sec/ng MLCK were significantly less in adult (0.26±0.01) than in fetal (1.52±0.11) arteries. In contrast, MLCK activities in adult (15.8±1.5) and fetal (17.3±1.3) artery homogenates were not significantly different. When in-situ fractional activation was calculated, adult values (1.72±0.17%) were significantly less than fetal values (9.08±0.83%). Together, these results indicate that decreased thick-filament reactivity in adult compared to fetal ovine carotid arteries is due at least in part to greater MLCK activity in fetal arteries, which in turn cannot be explained by differences in MLCK, MLC20 or calmodulin concentrations. Instead, this difference appears to involve age-related differences in fractional activation of the MLCK enzyme.