Effects of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) on isometric tension development and myosin light chain (P-LC) phosphorylation were determined in intact smooth muscle strips of bovine trachea. Addition of 10(-5) M carbachol resulted in a marked increase in the P-LC phosphorylation within 30 sec of stimulation. Subsequently, there was a gradual decrease in the P-LC phosphorylation. In contrast, steady-state contraction was not attained until 10 min of stimulation. In these strips, W-7 itself caused a contraction, probably due to the release of endogenous histamine that was blocked by pyrilamine. In the presence of W-7 and pyrilamine, the isometric tension development and P-LC phosphorylation during the initial 5-min stimulation period were inhibited. Concentrations of W-7 that caused a 50% inhibition were 145 and 125 microM, respectively, for tension and phosphorylation. N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), a weak calmodulin antagonist, produced no significant inhibition on the carbachol-induced isometric tension development and P-LC phosphorylation. After 10-min exposure to carbachol, W-7 inhibited the steady-state contraction without inhibition of the P-LC phosphorylation. These results suggest that 1) W-7 inhibits the smooth muscle contraction through the inhibition of the initial increase in the P-LC phosphorylation and 2) the maintenance of isometric tension (after 10 min exposure to carbachol) in unlikely to depend on the P-LC phosphorylation.