SummaryIncreased airway responsiveness occurs in normal young individuals compared to adults. A maturation of airway smooth muscle (ASM) contractility is likely a mechanism of this juvenile airway hyperresponsiveness. Indeed, we showed in guinea pig tracheal smooth muscle (TSM) that maximum shortening velocity decreases dramatically after the first 3 weeks of life. Because the phosphorylation of the 20-kDa myosin light chain (MLC 20 ) was shown to be a key event in ASM contractility, in the present work we sought to investigate it during ontogenesis. In three age groups (1-week-old, 3-week-old, and adult guinea pigs), we assessed the amount of MLC 20 phosphorylation achieved either in TSM crude protein homogenates exposed to Mg 2+ ·ATP·CaCl 2 or in tracheal strips during electrical field stimulation (EFS). Phosphorylated and unphosphorylated MLC 20 were separated on nondenaturing 10% polyacrylamide gels, and the ratio of phosphorylation was obtained by densitometric analysis of chemiluminescent Western immunoblots. Maximum MLC 20 phosphorylation (% of total MLC 20 ) in TSM tissue homogenate was, respectively, 32.6 ± 5.7, 32.2 ± 5.7, and 46.8 ± 5.8 in 1-week, 3-week, and adult guinea pigs. Interestingly, in nonstimulated intact tracheal strips, we found a substantial degree of MLC 20 phosphorylation: respectively, 42.2 ± 5.8, 36.5 ± 7.8, and 46.4 ± 4.7 in 1-week, 3-week, and adult guinea pigs. Maximal EFS-induced MLC 20 phosphorylation (% increase over baseline) in the 3-week age group was attained after 3 sec of EFS, and was 161.2 ± 17.6, while in 1-week and adult guinea pigs, it was attained at 1.5 sec of EFS and was, respectively, 133.3 ± 9.3 and 110.2 ± 3.9 (P<0.05). We conclude that MLC 20 phosphorylation in guinea pig intact tracheal strips correlates with ontogenetic changes in shortening velocity and changes in myosin light chain kinase content. These results further suggest that the maturation of ASM contractile properties plays a role in the greater airway responsiveness reported in children and young animals.