Ischemia produces striking electrophysiological abnormalities in blood-perfused hearts that may be caused, in part, by effects of ischemia on intracellular calcium. To test this hypothesis, intracellular Ca²⁺ concentration ([Ca²⁺]_i) transients were recorded from the epicardial surface of blood- and saline-perfused rabbit hearts using the long-wavelength indicator Fura Red. Calcium transients were much larger than the movement artifact, representing up to 29% of the total signal. Switching the perfusate from saline to blood did not affect the time course of the transients or the apparent level of [Ca²⁺]_i. Compartmentation of Fura Red fluorescence was estimated by exposure to Mn²⁺. The results were cytosol 60 ± 3%, organelles 12 ± 2%, and autofluorescence plus partly deesterified Fura Red 29 ± 4%. [Ca²⁺]_itransients were calibrated in situ by perfusion of the extracellular space with high-Ca²⁺ and Ca²⁺-free EGTA solutions. Peak systolic [Ca²⁺]_iwas 663 ± 74 nM, and end-diastolic [Ca²⁺]_iwas 279 ± 59 nm. Ischemia was produced by interruption of aortic perfusion for 2.5 min during pacing (150 beats/min). Ischemia produced broadening of the [Ca²⁺]_itransient, along with beat-to-beat alternations in the peak systolic and end-diastolic level of [Ca²⁺]_i(calcium transient alternans). [Ca²⁺]_itransient alternans occurred in 82% of blood-perfused hearts vs. 43% of saline-perfused hearts. The discrepancy between large and small transients (mean alternans ratio) was larger in the blood-perfused hearts (0.23 ± 0.04 vs. 0.07 ± 0.03,P = 0.005). These observations are important because of the apparent relationship of [Ca²⁺]_itransient alternans to electrical alternans and arrhythmias during ischemia.