The β-adrenergic antagonist, timolol, was previously shown to exert a protective effect in cats subjected to 5 h of myocardinal ischemia. The present study was designed to determine the effect of timolol on infarct size in cats 24 h after coronary occlusion. Timolol (25 μg/kg) or vehicle (0.9% NaCl) was administered 0.5, 5, 10, 15 and 20 h after acute ligation of the left anterior descending coronary artery. There was no significant difference in arterial blood pressure or heart rate in MI cats receiving timolol or vehicle. Timolol markedly decreased S-T segment elevation at 2–12 h (P<0.05). Left ventricular weights in MI+ vehicle cats (8.5±0.7g, n=6) were similar to timolol-treated MI cats (8.9±0.4 g, n=7). However, the percent of the left ventricular myocardium infarcted, determined by nitroblue tetrazolium staining, was significantly less (P<0.001) in timolol MI cats compared to saline-treated cats, 9.8±1.2% (n=7) vs 18.9±1.8% (n=6), respectively. Hemodynamic or cytoprotective actions of timolol do not appear to explain these results. Rather, the mechanism of infarct size reduction by timolol is probably explained by antagonism of β-receptor-mediated metabolic effects.