The hypothesis that rat cardiomyocytes become susceptible to hypercontracture after anoxia/reoxygenation was investigated. The cells were gradually overloaded with Ca2+ after different periods of simulated ischemia (substrate-free anoxia, medium at pH 6.4) followed by 20 minutes of reoxygenation. The cytosolic Ca2+ concentration (measured with fura 2) at which the cells developed maximal hypercontracture (Camax) was used as an index for their susceptibility to hypercontracture (SH). SH was increased in cardiomyocytes after prolonged periods of simulated ischemia; ie, these cells developed hypercontracture at significantly lower cytosolic Ca2+ levels than did normoxic cells (Camax, 0.80 +/- 0.05 micro mol/L versus 1.27 +/- 0.05 micro mol/L; P<.01). To find the possible cause of increased SH, the influence of Ca2+ overload, acidosis, and protein dephosphorylation were studied. Prevention of cytosolic Ca2+ overload in anoxic cardiomyocytes or imitation of ischemic acidosis in normoxic cells did not influence Camax. In contrast, use of 10 micro mol/L cantharidin (inhibitor of protein phosphatases 1 and 2A) during anoxic superfusion prevented the reduction of Camax. Furthermore, treatment of normoxic cardiomyocytes with 20 mmol/L of the chemical phosphatase 2,3-butanedione monoxime reduced Camax. Therefore, prolonged simulated ischemia increases susceptibility of cardiomyocytes to hypercontracture. This seems to be due to protein dephosphorylation. (Circ Res. 1997;80:69-75.)