Caffeine is a potent facilitator of Ca²⁺-induced Ca²⁺ release (CICR) and is widely used to study the role of ryanodine receptors (RyRs) in the control of smooth muscle function. Studies of the role of caffeine in intact tissues can be complicated to interpret if Ca²⁺ released by CICR also activates IP₃ receptors. We have therefore used guinea-pig ureter, which only expresses RyRs (1) to elucidate the effects of caffeine in intact smooth muscle. Adult guinea-pigs were humanely killed and the ureters dissected and loaded with Fluo-4 or Indo 1 (15μM) for 120 min at room temperature, and force and membrane potential recorded as previously described (2). Single cells were also produced by enzymatic digestion and voltage clamped. Data are means ± S.E.M.s, and n is number of animals or cells and is between 5 and 25. Significance was tested by t tests, taking significance at P<0.05. In intact ureteric muscle caffeine (1mM) produced selective inhibition of the plateau component of the action potential, and the amplitude and duration of the Ca²⁺ transient and phasic contractions. It also significantly increased the length of the refractory period from 40-90s to 3-4 min, when tested by the ability of the ureteric muscle to produce an action potential in response to suprathreshold depolarising pulses (3-5V, 100ms in duration). Confocal microscopy demonstrated that caffeine initiated Ca²⁺ sparks in both isolated cells and cells in situ. In voltage-clamped cells dialysed with the KCl pipette solution, spontaneous transient outward currents (STOCs) were elicited by caffeine, and it also significantly increased the ratio of outward to inward currents evoked by depolarising steps from a holding potential of -70 mV to 0 mV. Using the same protocol of stimulation but with CsCl in the pipette solution, caffeine had no effect on inward current but slightly potentiated Ca²⁺ transients evoked by depolarising voltage steps. Ryanodine (50μM) and cyclopiazonic acid (20μM) both inhibited Ca²⁺ sparks and reversed the inhibitory action of caffeine on the action potential and excitability. The inhibitory actions of caffeine on the action potential and excitability were also abolished by TEA (2mM) or iberiotoxin (200nM), blockers of Ca²⁺-activated K⁺ channels. These data suggest that in the guinea-pig ureter the inhibitory action of caffeine on the action potential and excitability is mediated by activation of a Ca²⁺ spark/STOCs coupled mechanism.