Reperfusion or reoxygenation of ischaemic or hypoxic cardiac tissue results in increases in total cell calcium and cell lysis both of which are dependent on mitochondrial function. Although changes which occur during this period of exposure of the tissue to hypoxia predispose the heart to take up calcium on reoxygenation, the mechanisms involved are not well understood. In the present study we have investigated the effects of hypoxia and reoxygenation on the concentration of intramitochondrial (matrix) free Ca2+ ([Ca2+](m)) using mitochondria loaded in the intact heart with fura-2. During periods of up to 80 min hypoxia, total tissue calcium content was unchanged. Over this period [Ca2+](m) rose from an initial value of 156±26 nM to 360±33 nM and 574±62 nM at 50 and 80 min of hypoxia, respectively; values that are within the expected physiological range for [Ca2+](m). Reoxygenation after 50 min hypoxia resulted in no further change in [Ca2+](m) whereas reoxygenation after 80 min hypoxia resulted in a 10-fold increase in this parameter. These results provide clear evidence that [Ca2+](m) increases during hypoxia and suggest that the ability of the cell to main Ca2+ homeostasis is lost on reoxygenation after prolonged hypoxia with the result that [Ca2+](m) exceeds the normal physiological range. © 1993 Academic Press Limited.