Cutaneous thermal injury resulting in burns covering approximately 45% of the total body surface area initiates metabolic alterations which contribute to subsequent myocardial dysfunction. Alterations in calcium homeostasis have been proposed as one mechanism by which burn injury alters organ function. This study used fura-2 and time-resolved single cell fluorescence microscopy to examine stress-related alterations in intracellular calcium in isolated adult rat cardiac myocytes. Ventricular myocytes were isolated from rats given a full-thickness scald burn comprising 43% of the total body surface area and fluid resuscitated with lactated Ringer's by the Parkland formula; control animals were included for comparison. Burn trauma caused a significant increase in cardiac myocyte maximal (peak systolic) and minimal (diastolic) mean cytosolic free calcium concentration ([Ca2+]i) transient ratios when compared to [Ca2+]i transient ratios measured in control rats. Isoproterenol application altered the time course of the [Ca2+]i transients of normal myocytes but this response was not observed in myocytes from the thermally injured rats. In addition, isoproterenol application to normal myocytes produced a significant increase in the amplitude of cell edge motion (+50%) compared to the cell edge motion measured in myocytes without isoproterenol stimulation; however, this cell motion response did not occur after isoproterenol application to myocytes from thermally injured rats. Caffeine application increased the maximal and minimal [Ca2+]i transient ratios of all myocytes, regardless of a burn injury, and the time course of the [Ca2+]i transients from the two groups appeared similar in the presence of caffeine as the myocytes progressed to contracture. Our data suggest that burn-mediated alterations in calcium homeostasis contribute, in part, to the cardiac contractile dysfunction which occurs after burn injury.