The early effects of metabolic inhibition on intracellular Ca²⁺ concentration ([Ca²⁺]_i), Ca²⁺ current, and sarcoplasmic reticulum (SR) Ca²⁺ content were studied in single pacemaker cells from the sinus venosus of the cane toad. The amplitude of the spontaneous elevations of systolic [Ca²⁺]_i (Ca²⁺ transients) was reduced after 5-min exposure to 2 mM NaCN from 338 ± 30 to 189 ± 37 nM (P < 0.005, n = 9), and the spontaneous firing rate was reduced from 27 ± 2 to 12 ± 4 beats/min (P < 0.002, n = 9). It has been proposed that CN acts by inhibition of cytochrome P-450, resulting in a reduction of cAMP and Ca²⁺ current. To test this proposal, we used clotrimazole, a cytochrome P-450 inhibitor, which also decreased the Ca²⁺ transients and firing rate. CN caused an insignificant fall of Ca²⁺ current (23 ± 11%) but a substantial reduction of SR Ca²⁺ content (by 65 ± 5%), whereas clotrimazole produced a larger reduction of Ca²⁺ current and did not affect the SR Ca²⁺ content. Thus the main effect of CN does not seem to be through inhibition of cytochrome P-450. In conclusion, CN appears to reduce Ca²⁺ release from the SR mainly by reducing SR Ca²⁺ content. A likely cause of the decreased SR content is reduced Ca²⁺ uptake by the SR pump.