We previously reported in guinea pig tracheal smooth muscle that maximal shortening velocity decreases from 3 weeks of age to adulthood. It is not known whether myosin light chain kinase (MLCK), a key enzyme determining the velocity of smooth muscle contraction, undergoes maturational changes. In the present work, we investigated MLCK protein content and mRNA expression in 1-week-old, 3-week-old, and adult guinea pigs. We extracted either proteins or RNA from isolated tracheal smooth muscle. The content of MLCK was assessed by Western immunoblots. MLCK mRNA was evaluated by Northern analysis and by quantitative real time reverse transcriptase-polymerase chain reaction (RT-PCR). The content of MLCK increased 3-fold at 3 weeks of age and then decreased in adults, being 0.116 +/- 0.042, 0.330 +/- 0.125 (P < 0.05), and 0.153 +/- 0.054 microg/mg of total protein, respectively, in 1-week, 3-week, and adult animals. Quantitative RT-PCR revealed that MLCK mRNA increased with age to 135 +/- 35% and 177 +/- 23% (P < 0.01) in 3-week and adult animals, respectively, compared to 1-week animals. The transient increase of MLCK content in juvenile guinea pig tracheal smooth muscle may contribute to the increased shortening velocity at this age. We suggest that this increased content of MLCK is one of the mechanisms leading to maturation of airway smooth muscle contractility, which in turn contributes to the airway hyperresponsiveness reported in children and young animals.