Regional myocardial blood flow (RMF) was measured simultaneously by use of labeled 8-mum microspheres and the constant-rate infusion of 3H2O in 22 open-chest dogs (Na pentobarbital anesthesia) under markedly different hemodynamic conditions. Following cardiac excision, three adjacent 80-mg tissue samples were taken from the subendocardial, mid-wall, and subepicardial layers of quadrantal left ventricular (LV) segments of the basal and midventricular cardiac slices, and from one segment of the apical slice, totaling 81 samples per LV. RMF was calculated by the microsphere reference-flow technique and by two 3H2O tissue-uptake models. Good agreement between techniques (r=0.9-0.96) was found in comparing flows to the myocardial layers. Using Kety's "single-mixer" model of 3H2O tissue uptake, fairly good agreement was found between techniques in the 80-mg tissue samples; the cofficient of variation from regression was 18.5% which improved markedly to 12.3% when the flow values for each technique were averaged in the three adjacent samples. Analysis of variance showed that flow to the various LV subdivisions (layer, segment, slice) of control animals was heterogeneous.