We previously described that carbachol (CCh) increased intracellular phosphatidic acid (PA) in guinea pig taenia coli and the increase was inhibited by diacylglycerol (DG) kinase inhibitor, R59022. The present study was conducted to clarify the role of DG kinase in CCh-induced increase in cytosolic free calcium and force development. Application of CCh to taenia coli induced transient increase in calcium level and contraction followed by sustained and lower calcium level and smaller contraction, and R59022 inhibited the latter responses specifically. The sustained responses were also evoked by 50 mM KCl, but these responses were slightly inhibited by R59022. Inhibition of calcium influx reduced responses to CCh but not KCl. Therefore, we consider that the sustained contraction induced by CCh is attributed to activation of DG kinase which generates PA and accelerates calcium influx.