The cardiotoxic effect of isoproterenol (ISO) is associated with, and possibly due to, calcium overload. Prior work suggests that calcium entry into cardiac myocytes after ISO administration occurs in two phases: an early rapid phase, followed by a slow phase beginning about 1 hour after ISO injection, leading to a peak myocardial calcium level after about 4 hours. We have tested the relationship of these phases to myocardial necrosis (MN) by determining the time after ISO administration at which the commitment to MN occurs. This was done by administration of propranolol at various times before and after ISO. In addition, since ISO induces lipolysis, and lipids can be toxic, experiments were conducted to determine if adrenergically-activated lipolysis could play a significant role in ISO-MN. We found that propranolol protected the myocardium equally well when administered anytime within 2 hours of ISO injection, but had no effect when given 4 hours after ISO. This showed that metabolic events taking place more than two hours after ISO injection are required for ISO-MN. As expected from prior work, there was a small and consistent amount of propranolol-resistant ISO-MN. Lipolysis, assessed by measuring serum glycerol levels, increased to tenfold above base line at one hour after ISO administration and returned to near basal levels at 4 hours. Potentiation of lipolysis by intravenous injections of phospholipase A2 (PLA2) or lipoprotein lipase (LPL) to rats treated with ISO substantially augmented MN. Propranolol completely blocked the increase in necrosis produced by PLA2 when given with ISO. Lipases induced only minimal necrosis in the absence of ISO. Administration of adenosine (an anti-lipolytic agent), oxfenicine (an inhibitor of mitochondrial palmitoyl carnitine transferase), or vitamin C (an anti-oxidant) resulted in a 55-60% reduction in MN. These results suggest that critical necrosis-determining events occur between 2 and 4 hours after ISO administration and imply a relationship between ISO-induced lipolysis, calcium influx, and ISO-MN. We hypothesize that importance of lipolysis as a determinant of ISO-MN is related to the generation of free fatty acids, their oxidized/metabolic products, or direct damage to plasma membrane.