Intracellular free calcium concentration ([Ca2+]i) was measured in isolated ferret ventricular papillary muscles during and after long exposures to ischemia. All experiments were performed at 37°C, and the muscles were stimulated at 1 Hz. Ischemia was simulated by changing from superfusion with oxygenated Tyrode's solution to superfusion with water-saturated gas (95% N2-5% CO2), thus simultaneously stopping oxygenation and restricting the extracellular space. [Ca2+]iwas measured with aequorin, which was microinjected into superficial cells of the preparation. Exposure to ischemia caused a complex series of changes in [Ca2+]i. In the first few minutes the changes in [Ca2+]iwere variable; however, after approximately 5 minutes all preparations exhibited a progressive increase in amplitude and duration of the stimulated rise in [Ca2+]i(the calcium transient). The amplitude of the calcium transients peaked after approximately 18 minutes of ischemia, when they were 339% of the control value. After this peak, the calcium transients progressively failed to occur in response to stimulation and declined in amplitude; simultaneously, spontaneous oscillations of [Ca2+]iappeared and increased in size and frequency. The oscillations in turn then gradually became less frequent until a large, prolonged (5–10 minute) increase in [Ca2+]ioccurred, after which [Ca2+]ireturned to a low level. There were no further oscillations after this event, which was seen on average after 37 minutes of ischemia. A slowly progressive contracture often began to develop at about this time. A gradual rise in resting [Ca2+]ioccurred during the remainder of the exposure to ischemia. When muscles were reperfused after long exposures to ischemia, there was a very large and prolonged increase in [Ca2+]i, which was usually associated with a contracture and failure of recovery of developed tension. The large increase in [Ca2+]icould be reduced by the inclusion of 3 mM nickel chloride in the reperfusing solution. Comparison between reperfusion with O2gas versus reperfusion with anoxic Tyrode's solution indicated that reoxygenation was more beneficial to the muscle than resumption of bulk flow. These results reveal the complex spectrum of changes in [Ca2+]ithat occur during ischemia and on reperfusion. These changes in [Ca2+]iare likely to play an important role in the generation of ischemic arrhythmias and muscle damage.