Background Acidosis-mediated injury to cardiac myocytes during surgery may lead to progressive heart failure. The nature of this injury, although not well defined, may be caused by induction of apoptosis in cardiac myocytes. We applied fluorescence imaging and biochemical techniques to assess apoptosis in cardiac myocytes excised from human patients and porcine subjects maintained on cardiopulmonary bypass to demonstrate the relationship between acidosis and apoptosis. Methods Multiphoton microscopy was used to image fluorescence signals generated in myocytes deep within atrial and ventricular biopsies for identification of apoptotic changes. The biopsies, obtained during cardiac surgery, were subjected to ex vivo or in vivo acidosis. Proapoptotic markers such as exposure of phosphatidyl serine, cytochrome c, apoptotic protease-activating factor-1, and caspase-3 were identified using fluorescence-based imaging and biochemical assays. Results Within 30 minutes of storage in low pH (<7) buffers, apoptosis was detected in human atrial samples, the severity of which correlated well with low pH. Apoptosis was also detected in atrial and ventricular biopsy samples obtained from three porcine subjects maintained on cardiopulmonary bypass and undergoing 110 minutes of aortic cross-clamp and 10 minutes of reperfusion, in which the cardiac pH was 6.36, 7.14, and 7.48. The apoptosis level detected in postacidotic reperfused cardiac tissue was pH dependent and approximately threefold greater than the precross-clamp levels. Conclusions Using fluorescence multiphoton microscopy and biochemical techniques we have assessed a direct correlation between low pH and induction of apoptosis in cardiac samples obtained both from human patients undergoing cardiac surgery and porcine subjects maintained on cardiopulmonary bypass simulating cardiac surgery.