Recent studies of the smooth muscle contractile system indicate that Ca(2+)-dependent phosphorylation of the 20-kDa myosin light chains, modulation of phosphoprotein phosphatases, and phosphorylation of thin-filament proteins are all potential features of contractile system regulation. The thin-filament proteins caldesmon and calponin are known to inhibit actomyosin ATPase in vitro and actin sliding velocity in the in vitro motility assay. Inhibition of actomyosin ATPase is relieved by phosphorylation of caldesmon or calponin. The notion that caldesmon and calponin phosphorylation-dephosphorylation is important in the living smooth muscle cell was tested using canine tracheal smooth muscle strips labeled with 32P. We found that both caldesmon and calponin phosphorylation increased in response to stimulation with carbachol. Carbachol induced a biphasic increase in [Ca2+]i in canine tracheal smooth muscle, an early transient increase in myosin phosphorylation, which decayed to 0.4 mol Pi/mol light chain, and a rapid transient increase in tissue shortening velocity. Relative changes in caldesmon phosphorylation correlate best with force development and the [Ca2+]i transient, both of which follow a similar time course. Calponin phosphorylation appears to be a rapid transient event more similar to the transient increase in unloaded shortening velocity. Our results are consistent with a potential role for both caldesmon and calponin phosphorylation in regulating smooth muscle contraction.