Simultaneous dual isotope (SDI) acquisition of (201)Tl rest/(99m)Tc-sestamibi stress-myocardial perfusion single photon emission computed tomography is a desirable new procedure in nuclear cardiology. In this protocol (201)Tl is injected at rest but imaging is performed not earlier than after exercise. Therefore, one must be convinced that throughout exercise (201)Tl remains distributed in an identical pattern as at rest. Before SDI can be applied clinically, (201)Tl rest MPS before and after exercise test needs to be compared for equality. The aim of this study was to assess the effect of time and exercise on the clearance of preinjected (201)Tl in normal and ischemic myocardium. In 122 patients rest (201)Tl and delayed (n =20) or poststress (n= 102) (201)Tl imaging was performed. Quantitative analysis of mean counts-per-pixel was performed for each segment in a 17-segment model. Differences between rest and delayed or poststress (201)Tl MPS were calculated. Patients with a poststress (201)Tl image were divided into normal (N= 66) and ischemic (N= 36) groups. Visual analysis was performed by two independent observers scoring the 17 segments on a scale of 0-4. The overall difference between rest (201)Tl and poststress (201)Tl MPS was - 15.4%. Normal and ischemic patients showed 16.2 and 14.0% (P =0.17) washout, respectively. Visual assessment by two independent observers revealed no regional differences between rest (201)Tl and delayed or poststress (201)Tl MPS. (201)Tl poststress MPS shows significant washout of thallium. This washout is not segmental, but global over the myocardium. No significant differences are found between normal and ischemic myocardium. The poststress (201)Tl MPS is a reliable reflection of rest perfusion. SDI acquisition of (201)Tl rest/(99m)Tc-sestamibi stress-MPS is clinically applicable.