A technique was developed and evaluated using the exponential infusion of positron-emitting diffusible tracers to quantitate myocardial perfusion. The approach employs a parameter that rapidly reaches a constant value as a function of tracer delivery rate, isotope decay constant, and the monotonically increasing tissue radioactivity. Isolated rabbit hearts with controlled flow were used to evaluate the approach, because tracer kinetics in such preparations mimic those in vivo. Accordingly, exponential infusions of H2 15O and [11C]butanol were administered to 25 isolated rabbit hearts perfused with Krebs-Henseleit solution (KH) alone or KH enriched with erythrocytes (KH-RBC, hematocrit = 40). With flow varied from 1.2 to 5 ml/g per min in eight KH hearts infused with H2 15O, actual and estimated flow correlated closely (r = 0.95, n = 52 determinations). For the KH-RBC hearts, flow was varied from 0.3 to 1.5 ml/g per min. Actual and estimated flow correlated significantly for both the 14 KH-RBC hearts infused with H2 15O (r = 0.90, n = 89 determinations) and the 3 KH-RBC hearts infused with [11C]butanol (r = 0.93, n = 13 determinations). In addition, the required exponentially increasing arterial tracer concentrations were shown to be attainable in vivo in dogs and rhesus monkeys after intravenous exponential administrations of tracer. The results suggest that the approach developed employing exponential tracer infusion permits accurate measurement of myocardial perfusion and that it should prove useful in the noninvasive measurement of regional myocardial perfusion in vivo by positron emission tomography.