Utilizing a model of chronic doxorubicin cardiomyopathy, this study examines the relationship between changes in expression and function of calcium handling proteins and contractile dysfunction. A possible mechanism to account for this relationship is suggested. New Zealand white rabbits were injected with either doxorubicin (1 mg/kg, twice weekly for 8 wk) or 0.9% NaCl. Gene transcript, protein levels, and the function of several proteins from the left ventricle were assessed. Protein levels of sarcoplasmic reticulum (SR) Ca2+ transporting ATPase (SERCA2a and b), Ca2+ release channel (RYR2), calsequestrin, Na/Ca exchanger, mRNA levels of RYR2, and [3H]-ryanodine binding (B(max)) to RYR2 were significantly decreased in doxorubicin-treated rabbits; protein levels of phospholamban, dihydropyridine receptor alpha2 subunit, and SR Ca2+ loading rates were not decreased. However, only protein levels of SERCA2 and RYR2, mRNA levels of RYR2, and Bmax of RYR2 significantly regressed with left-ventricular fractional shortening. Analysis of contractile function of atrial preparations isolated from doxorubicin-treated rabbits revealed that doxorubicin diminished contractility (dF/dt) of rest-potentiated contractions consistent with SR dysfunction. Serum concentrations of free triiodothyronine (T3) decreased in doxorubicin-treated rabbits. Our results suggest that chronic doxorubicin administration in the rabbit causes a SR-dependent contractile dysfunction that may result, in part, from decreased T3.