The effect of tert-butyl hydroperoxide (tBH) on the nucleotide level in isolated cardiomyocytes from adult rats was examined. Incubation of myocytes with the hydroperoxide resulted in loss of both ATP and NAD from mitochondria and cytosol. In contrast, only the cytosolic level of NADP was increased. The effect on myocyte content of ADP and AMP was less pronounced. A significant increase in formation and release of adenosine also occurred in tBH-exposed cells. Treatment of cardiomyocytes with tBH resulted in a progressive decline of rod-shaped and viable cells, which was accompanied by an increase in numbers of hypercontracted and round myocytes in the incubation mixture. Electron microscopic examination showed an intense contraction of the cells. In summary our results demonstrate that plasma membrane damage and hypercontraction are early manifestations of tBH-induced injury to isolated cardiomyocytes. The depletion of ATP and formation of adenosine also suggest that degradation of adenine nucleotides may contribute to the cytotoxicity caused by tBH. Possible mechanisms involved in the ATP depletion are discussed.