The authors compared the mortality and cardiac biomarker responses in three outbred stocks of Sprague Dawley rats (CD/IGS, Sasco, Harlan) treated with isoproterenol hydrochloride. Cardiac injury was confirmed by histologic evaluation, and increases in cardiac troponin I concentration in serum were measured by two methods. CD/IGS rats had a higher incidence and earlier mortality compared with Sasco or Harlan rats. Harlan rats had lower severity scores for cardiomyocyte degeneration/necrosis compared with the other stocks. Post-isoproterenol treatment cardiac troponin I concentrations were greater in CD/IGS and Sasco rats compared with Harlan rats. Concentrations of cardiac troponin T followed a similar pattern to that of cardiac troponin I in rats treated with isoproterenol. Myosin, light chain 3 concentrations increased in all rats treated with isoproterenol, but there was no difference between the three stocks in the magnitude or pattern of the dose response. Increases in fatty acid binding protein 3 concentrations were detected in only the highest dose group at the earliest timepoint postdose for all three stocks of rats. Results of these studies illustrate the need for investigators to recognize the potential differences in response between stocks of Sprague Dawley rats treated with cardiotoxicants or novel chemical entities.