We and others have shown that the fetal pulmonary arterial smooth muscle potential for contraction and relaxation is significantly reduced as compared with the adult. Whether these developmental changes relate to age-differences in the expression and/or activity of key enzymes regulating the smooth muscle mechanical properties has not been previously evaluated. Therefore, we studied the catalytic activities and expression of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) catalytic (PP1c) and regulatory (MYPT) subunits in late fetal, early newborn and adult rat intrapulmonary arterial tissues. In keeping with the greater force development and relaxation of adult pulmonary artery, Western blotting analysis showed that the MLCK, MYPT and PP1c content increased significantly with age and was highest in the adult rat. In contrast, their specific activities (activity/enzyme content) were significantly higher in the fetal as compared with the adult tissue. The fetal and newborn pulmonary arterial muscle relaxant response to the Rho-kinase inhibitor Y27632 was greater than the adult tissue. In addition to the 130 kDa isoform of MLCK, we documented the presence of minor higher molecular weight embryonic isoforms in the fetus and newborn. During fetal life the lung pulmonary arterial MLCK and MLCP specific activities are highest and appear to be related to Rho-kinase activation during lung morphogenesis.