Effects of stimulants and relaxants on the cytosolic Ca 2+ level ([Ca2+]cy,) and contraction were examined in isolated canine tracheal smooth muscle. High K + and carbachol induced a sustained increase in [CaZ+]cyt and muscle tension. Cumulative addition of KCI induced a graded increase in [Ca2+Jcy t and muscle tension. Cumulative addition of carbachol induced greater contraction than high K + at a given [Ca2+]cy t. 12-Deoxyphorbol 13-isobutyrate (DPB) (50 nmol/l) induced a small sustained contraction with little effect on [Ca2+]cy t. A higher concentration (1 #moll1) of DPB induced a larger sustained contraction with a decrease in [Ca2+]cyt. DPB (50 nmol/1) potentiated the KCl-induced contraction without or with only a small additional increase in [Ca2+]cy t. By contrast, 1/~mol/1 DPB potentiated the high-K+-induced contraction with a decrease in [Ca2+]cyt. Addition of 50 nmol/1 or 1 ,umol/1 DPB in the presence of carbachol inhibited both [Ca2+]cy t and muscle tension. Verapamil, isoprenaline and forskolin did not change or slightly decreased [Ca2+]cyt and muscle tension in resting trachea. Verapamil inhibited the contrction and [Ca2+]cy, stimulated by high K + and carbachol. Isoprenaline and forskolin inhibited the high-K+-induced contraction without changing [Ca2+]cyt, whereas these inhibitors inhibited carbachol-induced contraction with a relatively small decrease in [Ca2+]oy,. These results suggest that (a) sustained contractions induced by high K Β§ and carbachol are due to the sustained increase in [Ca2+]cyt, (b) carbachol increases the sensitivity of contractile elements to Ca 2+, and (c) isoprenaline and forskolin inhibit the contraction by the decrease in [Ca2+]cy t and also by the decrease in the sensitivity of contractile elements to Ca 2+. The carbachol-induced Ca 2 Β§ sensitization might be attributable to the activation of protein kinase C following the stimulation of the phosphatidylinositol turnover.