Inasmuch as smooth muscle contractile protein abnormalities may account for the maintenance of a high pulmonary vascular resistance, we evaluated the pulmonary arterial myosin light chain kinase (MLCK) and phosphatase (MLCP) in normal and pulmonary hypertensive (PH) fetal sheep. In addition, aorta and vena cava MLCP and MLCK activities were also measured. The MLCK activity(nanomoles/min/mg) was determined by the incorporation of[32P]PO4-3to the 20-kD smooth muscle myosin light chains and the MLCP activity by assaying for the dephosphorylation of the 20-kD myosin light chain (MLCP-light chain) and heavy meromyosin (MLCP-HMM). The MLCP content was determined by Western blot analysis. PH was characterized by a significant increase in the right-to-left ventricular wall weight ratio from 0.99 ± 0.04 in the control to 1.52 ± 0.12 in the experimental group (p< 0.01). The pulmonary MLCP-light chain and MLCP-HMM activities in the experimental group were 2.0 ± 0.2 and 1.3 ± 0.2 and significantly lower than in the control group values (3.8 ± 0.5 and 2.5 ± 0.3;p< 0.01). The MLCK activity was 9.6± 1.2 in the control and 7.8 ± 0.7 in the experimental fetal pulmonary artery (p= NS). The activities of both enzymes in the aorta and vena cava samples were not altered by PH. The MLCP content in experimental animals (0.50 ± 0.09 OD × mm2) was significantly lower than that for the control pulmonary tissue (1.72 ± 0.42;p< 0.01), suggesting that PH down-regulates pulmonary vascular MLCP expression. In conclusion, the maintenance of a high pulmonary vascular resistance in PH may be secondary to abnormalities in tissue content and/or activity of MLCP.Abbreviations: PPHN,persistent pulmonary hypertension syndrome of newborn;MLCK,myosin light chain kinase;MLCP,myosin light chain phosphatase;HMM,heavy meromyosin;PP1,PP2, protein phosphatase types 1 and 2;PP1M,myosin protein phosphatase;SMP,smooth muscle phosphatase