The rate-limiting enzyme for vascular contraction, Myosin Light Chain Kinase (MLCK), phosphorylates regulatory myosin light chain (MLC20) at rates that appear faster despite lower MLCK abundance in fetal compared to adult arteries. This study explores the hypothesis that greater apparent tissue activity of MLCK in fetal arteries is due to age-dependent differences in intracellular distribution of MLCK in relation to MLC20. Under optimal conditions, common carotid artery homogenates from non-pregnant adult female sheep and near-term fetuses exhibited similar values of Vmax and Km for MLCK. A custom-designed, computer-controlled apparatus enabled electrical stimulation and high-speed freezing of arterial segments at exactly 0, 1, 2, and 3 seconds, calculation of in situ rates of MLC20 phosphorylation, and measurement of time-dependent colocalization between MLCK and MLC20. The in situ rate of MLC20 phosphorylation divided by total MLCK abundance averaged to values more than 147% greater in fetal (1.06 ± 0.28) than adult (0.43 ± 0.08) arteries, which corresponded respectively to 43±10% and 31±3% of the Vmax values measured in homogenates. Confocal colocalization analysis revealed in fetal and adult arteries that 33 ± 6% and 20 ± 5% of total MLCK colocalized with pMLC20, and that MLCK activation was greater in peri-luminal than peri-adventitial regions over the time-course of electrical stimulation in both age groups. Together, these results demonstrate that the catalytic activity of MLCK is similar in fetal and adult arteries, but that the fraction of total MLCK in the functional compartment involved in contraction is significantly greater in fetal than adult arteries.