Cardiotoxicity associated with doxorubicin (DOX) treatment limits the therapeutic efficiency of this drug against cancer. 2-Chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide (Cl-IB-MECA), a selective agonist of A(3) adenosine receptor (A(3)R), reduces DOX toxicity in newborn rat cultured cardiomyocytes. The study's aim was to determine whether the protection demonstrated by Cl-IB-MECA attenuates cardiac depression in vivo. In addition, we wished to examine whether this protective pathway affects the sarcoplasmic reticulum (SR) calcium uptake and release, as well as intramitochondrial Ca(2+) accumulation induced by DOX. Rats were injected every alternate day (6 times) with (1) saline, (2) 2.5mg/kg i.p. DOX, (3) 33 microg/kg i.v. Cl-IB-MECA, (4) DOX+Cl-IB-MECA. Left ventricular functions were assessed by invasive (pressure) and non-invasive (echocardiography) techniques at the end of the injection period and 4 weeks later. Cytosolic and intramitochondrial calcium levels were measured with indo-1 and rhod-2 probes. SR Ca(2+) content was determined by exposing cultured rat cardiomyocytes to caffeine. Echocardiography data demonstrate left ventricular wall thinning (23%), an increase in the end systolic dimension (170%) and decreased fractional shortening (35+/-5% vs. 54+/-5%, p<0.01) in DOX-treated animals, compared to the control group. DOX increased Ca(2+) levels in the cytosol and in mitochondria by diminishing the SR Ca(2+) uptake. Pretreatment with Cl-IB-MECA attenuated left ventricular dysfunction, improved SR calcium storage capacity and prevented mitochondrial Ca(2+) overload. We conclude that the adenosine A(3) receptor agonist is effective in vivo against DOX cardiotoxicity via the restoration of Ca(2+) homeostasis and prevention of mitochondrial damage that occurs as a result of Ca(2+) overload.