Anthracyclines cause severe irreversible cardiac toxicity. The study of changes in cardiac permeability with chemotherapy could enhance the understanding of mechanisms behind cardiac damage, and provide useful information to evaluate anthracycline cardiotoxicity. Thirty-six rats (12 Sprague-Dawley, 12 Wistar, 12 Fischer-344) were randomly assigned to control (n = 21) or doxorubicin (n = 15), and injected i.p. with a cumulative dose of 18 mg/kg doxorubicin in saline (vehicle) or vehicle alone over 12 days. Echocardiography was performed at baseline and on day 11. An isolated heart experiment was done on day 12 to obtain perfused heart pressure values, and to measure cardiac capillary permeability using a Texas Red/sodium fluorescein multiple indicator dilution method. Control animals had significantly lower average permeability-surface-area-products (0.035 ± 0.013 cm(3)/s) than doxorubicin animals (0.066 ± 0.023 cm(3)/s), PSP ± SD, p < 0.001. These permeability changes correlated with significant functional changes. There was a significant decline in cardiac function with a deleterious effect of chemotherapy on fractional shortening (p < 0.001), left ventricular developed pressure (p < 0.001), contractility (p < 0.001), and relaxation (p = 0.02). Based on our results, cardiac capillary permeability changes can be detected after in vivo chemotherapy treatment using our fluorescent multiple indicator dilution technique, and may provide valuable information in evaluating cardiotoxicity of novel drugs.