1. The mechanism of the inhibitory effect of caffeine was investigated using vascular smooth muscle of rabbit aorta and intestinal smooth muscle of taenia isolated from guinea-pig caecum. 2. Caffeine, 0.5-10 mM, relaxed the sustained contraction induced by 65.4 mM KCl or 10(-6) M noradrenaline in aorta, and by 45.4 mM KCl or 10(-6) M carbachol in taenia. The inhibitory effect of caffeine on the high K+-induced contraction was antagonized by external Ca2+ but not by the Ca2 channel activators, Bay K 8644 (10(-7) M) or CGP 28,392 (10(-7) M). Forskolin (2 x 10(-7) M) potentiated the inhibitory effect of caffeine on the noradrenaline-induced contraction but not on the high K+- or carbachol-induced contraction. Caffeine induced a time- and concentration-dependent increase in the cyclic AMP content of aorta and forskolin caused a further augmentation. 3. 45Ca2+ uptake was increased by high K+ or noradrenaline in aorta and by high K+ or carbachol in taenia. The increments were inhibited by caffeine at concentrations needed to inhibit muscle contractions. 4. 45Ca2+ in the cellular releasable site in aorta was decreased either by noradrenaline or by caffeine. Simultaneous application of noradrenaline and caffeine did not induce an additive decrease. 5. In aorta treated with a Ca2+-free solution, caffeine induced only a small contraction. Noradrenaline induced a greater contraction which was inhibited by caffeine. After washout of caffeine and noradrenaline, the second application of noradrenaline induced a transient contraction suggesting that caffeine does not deplete the noradrenaline-sensitive store. 6. It was concluded that caffeine has multiple sites of action in smooth muscle. Caffeine releases Ca2+ from a store which is apparently not sensitive to noradrenaline. Caffeine may inhibit noradrenalineinduced Ca2' release. Caffeine itself induces only a small contraction possibly because it decreases the Ca2+ sensitivity of contractile filaments and/or increases Ca2+ extrusion. Further, caffeine seems to inhibit stimulated Ca2+ influx. Cyclic AMP may be only partly responsible for the inhibitory effect of caffeine.