Oxidative stress may alter cardiac function by affecting intracellular free Zn2+ concentrations ([Zn2+](i)). Rabbit ventricular myocytes loaded with fura 2 were used to fluorometrically measure resting [Zn2+](i) (0.23 ± 0.03 nM) and intracellular Ca2+ concentration ([Ca2+](i)) (36 ± 7 nM). Fluorescence quenching by the heavy metal chelator N,N,N',N'-tetrakis(2- pyridylmethyl)ethylenediamine was used to quantitate [Zn2+](i). The thiol- reactive oxidants hypochlorous acid (0.1 mM) and selenite (1 mM) increased [Zn2+](i) to 7.7 ± 1.7 and 6.1 ± 1.7 nM, respectively, within 5 min. Dithiothreitol (0.5 mM), a disulfide-reducing agent, rapidly restored normal [Zn2+](i). The oxidants did not affect [Ca2+](i). However, depolarization-induced Ca2+ transients and Ca2+ currents were zinc dependent. [Zn2+](i)-associated fluorescence was substantial and, if ignored, it led to overestimation of [Ca2+](i) by approximately twofold before oxidant treatment and by approximately eightfold after oxidants. The results demonstrate that [Zn2+](i) 1) can be greatly increased by thiol- reactive oxidants; 2) may contribute to oxidant-induced alterations of excitation-contraction coupling; and 3) has strong fura 2 fluorescence which, if overlooked, can lead to significant overestimation of [Ca2+](i).