At near-threshold substance P concentrations, the isometric tension response of bovine tracheal strips is almost completely abolished by atropine, indicating mediation of contraction via substance P-stimulated release of acetylcholine from prejunctional nerve terminals. At near-maximal concentrations, the atropine-inhibited component of the tension response is less than 25%, indicating mainly direct activation. Under conditions in which activation by substance P is direct, peak tension is reached in approximately 11 min. Immunoblot analysis of the time course of phosphorylation of the 20-kDa myosin light chain (LC20) reveals incorporation of approximately 0.5 mol phosphate/mol light chain at 10 min. Two-dimensional tryptic phosphopeptide analysis of phosphorylated light chain reveals a single major phosphopeptide. The peptide migrates identically with that produced by myosin light chain kinase phosphorylation of purified tracheal myosin in vitro. Contraction stimulated by acetylcholine is more rapid, with attainment of peak tension in 2.5 min and a peak LC20 phosphorylation of 0.65 mol/mol. These results indicate that 1) substance P mediates contraction of bovine trachea both directly and indirectly, and 2) under conditions in which activation is direct, the tension and phosphorylation responses qualitatively resemble those observed with acetylcholine.