Summary: To investigate the role of cellular proteinases in acute myocardial infarction, we measured the rate of total proteolysis (ie, degradation of proteins to amino acids and oligopeptides) in slices obtained from nonischaemic myocardium, 1 h ischaemic endocardium, 3 h ischaemic epicardium, and 3 h ischaemic endocardium in open-chest dogs. Slices were incubated either in high pO2 or in low pO2 Krebs-Ringer-bicarbonate buffer with cycloheximide; proteolysis was estimated from tyrosine production. Tyrosine production in each of the ischaemic tissues incubated in low pO2 medium was lower than that in nonischaemic myocardium incubated in high pO2 medium. This suggests that proteolysis is inhibited during acute myocardial infarction both before (3 h ischaemic epicardium) and shortly after (1 h ischaemic endocardium, 3 h ischaemic endocardium) irreversible damage is believed to occur. Tyrosine production in 1 h and 3 h ischaemic endocardium was similar to that in 3 h ischaemic epicardium, indicating that no increase in proteolytic activity accompanies the progression of ischaemic damage from the phase of reversibility to that of presumed irreversibility. Ischaemic tissues did not exhibit a rebound increase in proteolysis when incubated in high pO2 medium, suggesting that reperfusion of acutely ischaemic myocardium is not associated with activation of proteolytic enzymes. Leupeptin, an inhibitor of thiol-proteinases, produced similar decreases of proteolysis in nonischaemic and ischaemic myocardium: thus, lysosomal thiol-proteinases (cathepsins B, L and H) do not appear to be activated in acute myocardial ischaemia. The data provide evidence that total proteolysis decreases throughout the phase of acute myocardial infarction during which irreversible injury develops, suggesting that cellular proteinases may not play a causal role in ischaemic myocellular death.