Background Myocardial uptake of either Tl-201 or Tc-99m sestamibi (sestamibi) is known to plateau at high coronary flow rates. However, few direct comparisons have been made between these tracers to determine what effect differences in the uptake plateau for the two tracers may have on the detection of coronary stenoses of various severities. Methods and Results Twenty-two dogs were instrumented with flow transducers on the left anterior descending (LAD) and circumflex (LCx) arteries. In 6 nonstenotic dogs, adenosine was infused directly into the LAD, whereas 16 dogs with either critical (n=7) or mild (n=9) LAD stenoses received an intravenous infusion. At peak flow, Tl-201 (0.5 mCi), sestamibi (5 to 8 mCi), and radiolabeled microspheres were injected simultaneously. Five minutes later, dogs were killed, and ex vivo imaging of heart slices and gamma-well counting of multiple myocardial samples was performed. Neither Tl-201 nor sestamibi uptake increased in direct proportion to flow. In the 6 nonste notic dogs, a fivefold increase in LAD flow increased Tl-201 and sestamibi uptake by only 202+/-6% and 138+/-4%, respectively (P<.0001). In the dogs with critical stenosis, the ratios of stenotic to normal activity by well counting for Tl-201 (0.37+/-0.05) and sestamibi (0.53+/-0.06) underestimated the microsphere-determined flow disparity (0.17+/-0.03) (P<.005), but the degree of underestimation was greater for sestamibi (P=.001). Similarly, in the dogs with mild stenosis, the stenotic-to-normal ratio for Tl-201 (0.62+/-0.04) approximated the flow ratio (0.43+/-0.04) better than sestamibi (0.79+/-0.03) (P<.0001). Sestamibi defects, however, were visually identifiable on the images of the myocardial slices. By image quantification, sestamibi defect magnitude (LAD-to-LCx count ratio) in the critical stenosis group (0.62+/-0.05) was significantly less than in the mild stenosis group (0.80+/-0.02) (P<.01). Conclusions Thus, with adenosine-induced hyperemic flow, both Tl-201 and sestamibi significantly underestimated the magnitude of the flow disparity between stenotic and normal perfusion beds. The degree of underestimation was greater for sestamibi. The clinical implication of these experimental findings for vasodilator perfusion imaging remains to be determined, since factors such as greater redistribution and scatter with Tl-201 could offset its advantages.