In this study we aimed to determine the concentration range of cyclosporin A (CsA) which was effective in protecting against reoxygenation injury in isolated cardiomyocytes, and its effects on intramitochondrial free calcium levels ([Ca2+]m). We also determined whether a high [CsA] had any deleterious effect on normal myocyte function. Isolated adult rat ventricular myocytes were placed in a chamber on the stage of a fluorescence microscope for induction of hypoxia. [Ca2+]m was determined from indo-1/am loaded cells where the cytosolic fluorescence signal had been quenched by superfusion with Mn2+. Cell length was measured using an edge-tracking device. Upon induction of hypoxia, control cells underwent rigor-contracture in 37 +/- 1 min (n = 99) (T1); CsA had no effect on T1. The percentage of control cells which recovered upon reoxygenation depended on the time spent in rigor (T2). With a T2 of 21-30 min, only 36% of control cells recovered compared with 90% and 78% of cells treated with 0.2 microM and 1 microM CsA respectively. After 40 min in rigor, [Ca2+]m was 280 +/- 60 nM in control-recovered cells (50% of cells) and 543 +/- 172 nM and 153 +/- 26 nM in cells treated with 0.2 and 1 microM CsA, respectively (all CsA treated cells recovered). In normoxic studies, CsA had no effect on cell contractility or [Ca2+]m upon rapid pacing, even in presence of an elevated external [Ca2+]. In conclusion, both low and high [CsA] protected against reoxygenation injury to cardiomyocytes despite having opposing effects on [Ca2+]m, suggesting more than one mechanism of action. CsA had no effect on either cell contractility or [Ca2+]m in normoxic cells.