The normally positive force- and Ca2+-frequency responses (FFR and CaFR) are inverted in heart failure (HF); whether oxidative stress contributes to these abnormalities is unknown. We evaluated the impact of acute and prolonged oxidative stress on contraction and Ca2+ handling in adult rat cardiomyocytes. Acute (30:min) exposure to H2O2 (100 μM) induced a twofold increase (P < 0.025) in intracellular oxyradicals together with contractile depression despite preservation of the Ca2+ transient and the FFR and CaFR to 3:Hz, indicating reduced myofilament Ca2+ responsiveness. In contrast, prolonged (24:h) exposure to the copper-zinc superoxide dismutase inhibitor diethyldithiocarbamic acid (DDC, 1 μM) similarly augmented oxyradicals but also increased cell size, and contraction and Ca2+ transient duration (P < 0.025). DDC-treated myocytes displayed inverted FFRs and attenuated (though still positive) CaFRs as compared to control, indicating reduced myofilament Ca 2+ responsiveness coupled with altered Ca2+ handling. Protein levels of the Na+-Ca2+ exchanger (NCX), sarcoplasmic reticular (SR) Ca2+ ATPase (SERCA2), and serine-16 phosphorylated phospholamban (pSer16-PLB) were increased (P < 0.025), whereas dihydropyridine receptor abundance was decreased. Total PLB and ryanodine receptor protein expression were unchanged. Caffeine-induced Ca2+ release showed increased NCX activity (P < 0.025) without changes in total releasable SR Ca2+, suggesting compensatory changes in SERCA2 and pSer16-PLB to maintain SR Ca2+ load. The superoxide scavenger Tiron attenuated these effects. Thus, acute oxyradical exposure rapidly depresses myofibrillar Ca2+ responsiveness. Prolonged oxidative stress further induces alterations in Ca2+ handling that combined with extant reductions in myofibrillar responsiveness invert the FFR. With regard to Ca 2+ handling, reduced transsarcolemmal Ca2+ flux rather than reduced SR Ca2+ uptake was the primary determinant of a negative FFR. Analogous changes may be operative in HF, a state characterized by both oxidative stress and Ca2+ dysregulation. © 2005 Elsevier Ltd. All rights reserved.