We have previously reported that contractile reactivity of human airway preparations in vitro depends on sensitization status. The aim of this study was to examine whether this could be associated with differences in the content and/or expression pattern of myosin light-chain kinase (MLCK) isoforms in airway smooth muscle (ASM). Macroscopically normal lung tissue was obtained from subjects undergoing lung transplantation, and smooth-muscle bundles were dissected from nonsensitized (n = 5) and sensitized (n = 5) bronchi. MLCK isoform expression was then assessed by immunoblotting. The major MLCK isoform in ASM was smooth-muscle MLCK (smMLCK; 136 kD). Nonmuscle MLCK isoforms (nmMLCK; 210 to 220 kD) were not present. The smMLCK content was significantly higher in ASM from sensitized bronchi (p = 0.049) than in ASM from nonsensitized tissue (11.9 +/- 3.3 versus 4.1 +/- 0.7 arbitrary units [a.u.] smMLCK/mg ASM, respectively). In contrast, there was no significant difference (p = 0.636) in the content of myosin heavy chain (MHC) in tissue collected from sensitized and nonsensitized bronchi (1.33 +/- 0.33 versus 1.09 +/- 0.37 microg MHC/mg ASM, respectively). This study is the first to examine MLCK isoforms in human ASM, and suggests that increased smMLCK content may be one of the mechanisms responsible for enhanced contractile reactivity in sensitized tissue.