Responses of isolated adult rat heart cells to conditions that emphasize various aspects of ischemia have been evaluated. Cells maintained in hypoxic media with limited gubstrate deteriorate mort rapidly than aerobic controls supplemented with glucose. Two distinct irreversible pathways for cell alteration can be distinguished as follows: (1) continued anaerobic aging in the absence of glucose results in the production of large numbers of cells which retain the rod-shaped morphology of heart cells in situ, but which have lost saxcolemmal integrity, and (2) after a period of anaerobic aging, reaeration of the cells produces large numbers of rounded cells in irreversible contracture. These cells maintain an intact sarcolemma and are indistinguishable from those produced by addition of 1 mM Ca2+to Na+-loaded, aerobic cells. Contracture of isolated cells on re-aeration is at least superficially analogous to the oxygen paradox in situ, but since the isolated cells maintain an intact sarcolemma, there is no loss of creatine phosphokinase or other components of the cytosol. Incubation of isolated heart cells at acid pH (pH 8.8 to 6.2) largely prevents both Ca2+-dependent contracture and a Ca2+- dependent loss of respiratory capacity. The acidic conditions virtually eliminate the net influx of40Ca2+into isolated cells that occurs at neutral pH, and the inhibition appears to be localized at the sarcolemma.