Caffeine is thought to affect excitation-contraction coupling in cardiac muscle by activating the sarcoplasmic reticulum (SR) Ca2+-release channel. The effect of caffeine at the single channel level was studied by incorporating canine cardiac SR vesicles into planar lipid bilayers. Cardiac Ca2+-release channels were activated in a steady-state manner by millimolar cis-caffeine and displayed a unitary conductance (77 pS in 50 mM Ca2+ trans) similar to that previously observed for the Ca2+-activated cardiac channel. The caffeine-activated channel was moderately sensitive to the voltage applied across the bilayer, was sensitive to further activation by ATP, and was inhibited by Mg2+ and ruthenium red. Kinetic analysis showed that at low Ca2+ concentration, caffeine activated the channel by increasing the frequency and the duration of open events.