We sought to understand the effect of a transient exposure of cardiac myocytes to H₂O₂ at a concentration that did not induce apoptosis. Myocytes were exposed to 30 µmol/L H₂O₂ for 5 minutes followed by 10 U/mL catalase for 5 minutes to degrade the H₂O₂. Cellular superoxide was measured using dihydroethidium. Transient exposure to H₂O₂ caused a 66.4% increase in dihydroethidium signal compared with controls exposed to only catalase, without activation of caspase 3 or evidence of necrosis. The increase in dihydroethidium signal was attenuated by the mitochondrial inhibitors myxothiazol or carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone and when calcium uptake by the mitochondria was inhibited with Ru360. We investigated the L-type Ca²⁺ channel (I_Ca-L) as a source of calcium influx. Nisoldipine, an inhibitor of I_Ca-L, attenuated the increase in superoxide. Basal channel activity increased from 5.4 to 8.9 pA/pF. Diastolic calcium was significantly increased in quiescent and contracting myocytes after H₂O₂. The response of I_Ca-L to -adrenergic receptor stimulation was used as a functional reporter because decreasing intracellular H₂O₂ alters the sensitivity of I_Ca-L to isoproterenol. H₂O₂ increased the K_0.5 required for activation of I_Ca-L by isoproterenol from 5.8 to 27.8 nmol/L. This effect and the increase in basal current density persisted for several hours after H₂O₂. We propose that extracellular H₂O₂ is associated with an increase in superoxide from the mitochondria caused by an increase in Ca²⁺ influx from I_Ca-L. The effect persists because a positive feedback exists among increased basal channel activity, elevated intracellular calcium, and superoxide production by the mitochondria.