Aminothiol amifostine (AMI) protects against toxic effects of both ionizing radiation and numerous anticancer drugs. The aim of this study was to investigate the potential protective effects of AMI against doxorubicin (DOX)-induced cardiotoxicity in rats. Male Wistar rats were treated with AMI (75 mg/kg i.p.) and/or DOX (1.25 mg/kg i.p.), 4 times per week, for 4 weeks. Mortality, general condition and body weight of the animals were observed during the whole treatment, and for a further 4 weeks, until the end of experiment. Evaluation of cardioprotective efficacy of AMI was performed by analyzing the electrocardiographic parameters and response to the pro-arrhythmic agent aconitine, as well as activity registration of the in situ rat heart preparations. Necropsy was also performed at the end of the experiment, and heart excision, weight and macroscopic examination were done before histological evaluation. Doxorubicin caused rat heart disturbances manifested by prominent electrocardiographic changes (Salpha-T prolongation and T-wave flattening), significantly enhanced response to aconitine, decrease of the heart rate and contractility, as well as histopathologically verified myocardial lesions. The heart changes were accompanied by 40% mortality rate, significant decline in body mass and severe effusion intensity score in 66.6% of the animals. Application of AMI before each dose of DOX significantly reduced or completely prevented its toxic effects. Therefore, since AMI had very good protective effects against a high dose of DOX given as a multiple, low, unitary dose regimen, not only on the heart but on the whole rat as well, it could be recommended for further investigation in this potentially new indication for clinical application.