The microsphere technique has been used to follow changes in collateral blood flow as a function of time and to relate acute changes in collateral flow to subsequent myocardial necrosis after coronary occlusion. However, flow studies in chronic preparations are complicated by an apparent loss of microspheres from necrotic myocardium. Preocclusion or control flow measured 2-4 days after coronary occlusion is reduced in infarcted compared with nonischemic areas. This apparent reduction in flow could result either from actual loss of microspheres from the infarct or from increased tissue weight. The latter possibility was examined by evaluating tissue components that could cause infarct expansion, i.e., water content and exogenous cellular elements (hemorrhage and inflammation), in 4- and 28-day-old infarcts produced by ligation of the circumflex artery in dogs. Infarct size measured at 4 days averaged 41% of the left ventricle (LV). Preocclusion blood flow (in ml/min/g) measured at 4 days was variably reduced in the infarcted circumflex (LCC) bed compared with the anterior descending (LAD) bed. Preocclusion LCC/LAD flow ratios ranged from 0.43-0.94. Analysis of these infarcts showed a 25% increase in infarct water content and extensive hemorrhage or acute inflammation or both, which added cellular elements to the infarcts; the severity of the latter was inversely proportional to the reduction in the LCC/LAD flow ratio. Thus, the decreased LCC/LAD ratios could be explained largely, if not entirely, by infarct expansion due to the addition of edema, hemorrhage and inflammation. By contrast, 28 days after coronary ligation, necrotic myocardium was largely replaced by dense scar tissue occupying only 11% of the LV. Contraction of these infarcts was associated with concentration of microspheres such that the LCC/LAD ratio was increased to 3.39. Thus, the changing composition of an evolving infarct results in a changing reference base. Early expansion of infarct volume due to edema and cellular infiltration causes an underestimation of collateral blood flow and an overestimation of infarct size when the data are referred either to tissue wet or dry weight. Later, when the infarct has been replaced by a contracted scar, directionally opposite errors occur. More accurate measurements of anatomic parameters or collateral flow can be achieved by using the preocclusion LCC/LAD flow ratio, as an index of the change in reference base, to calculate corrected anatomic and flow data.