Activation of sarcolemmal Ca²⁺-activated channels was investigated in single, voltage-clamped myocytes from the portal veins of guinea pigs (∼ 500 g, humanely killed by stunning then immediate exsanguination). Increases in cytosolic Ca²⁺ ([Ca²⁺]_c) were produced by photolysed caged IP₃ (25 µM) or by pressure ejection of caffeine (10 mM). At −70 mV, IP₃-evoked Ca²⁺ release induced an inward current with properties similar to a Ca²⁺-activated Cl^–current. These included activation by an increase in [Ca²⁺]_c,a reversal potential (−31 ± 4 mV, n=8, mean ± sem) close to the predicted equilibrium potential for Cl^–(−29 mV) and inhibition of the current by niflumic acid (100 µM; at −70 mV control − 565 ± 94 pA, n=9 and in niflumic acid −48 ± 39 pA, n=5, p<0.01, statistical significance was determined by unpaired Student’s t-test, where p<0.05 was considered significant). In the absence of extracellular Ca²⁺,IP₃ evoked neither a rise in [Ca²⁺]_c nor an inward current, confirming that the current did not arise from a direct action of IPon the channel. In contrast to IP₃,[Ca²⁺]_c increases evoked by RyR activation with caffeine did not activate a Ca²⁺-activated Cl^–current. Activation of Ca²⁺-activated K⁺ channels occurred at membrane potentials positive to ∼−25 mV, as monitored by spontaneous transient outward currents (STOCS). Caffeine and IP₃ each caused large outward Ca²⁺-activated K⁺ currents and subsequently inhibited STOCS suggesting that their actions were mediated by depleting SR Ca²⁺ stores. Guinea pig portal vein cells have two separate intracellular Ca²⁺ stores, one with both IP₃ receptors (IP₃R) and ryanodine receptors (RyR), and the other with RyR alone. The common store (IP₃R and RyR) requires extracellular Ca²⁺ for refilling; the store with RyR alone can refill in the absence of extracellular Ca²⁺. Removal of extracellular Ca²⁺ abolished STOCS but responses to caffeine were maintained suggesting that the store with RyR alone contained Ca²⁺ but did not contribute to the generation of STOCS. The selective activation of sarcolemmal Ca²⁺-activated channels by Ca²⁺ release from the common store (IP₃R and RyR) suggests this store is located peripherally, whereas the solely RyR store may be deeper in the myoplasm.