Beta-blocker therapy is used to decrease myocardial ischemia during exercise but may cause suboptimal diagnostic performance in exercise stress testing. The aim of the present study was to compare results of quantitative technetium-99-sestamibi single photon emission tomography (SPECT), following exercise stress test or pharmacological stress test with adenosine. We chose adenosine as comparison, since betablockers may not interfere with adenosine induced vasodilatation and therefore possibly may not interfere with its diagnostic performance. Sixteen patients with angiographically documented coronary disease (5 single-vessel, 6 two-vessel and 5 three-vessel disease), who were chronically treated with beta-blockers, performed SPECT imaging at rest, following bicycle exercise and following adenosine infusion in random order. The SPECT data were analyzed visually and quantitatively, using dedicated computer software (CEqual). According to both visual and quantitative SPECT analysis, adenosine was superior to show reversibility. Higher reversibility extent (50 ± 15 vs. 26 ± 12 pixels, p < 0.01) and more intense reversibility severity (110 ± 29 vs. 49 ± 23 sum of SDs, p < 0.05) were observed during adenosine than exercise. Conclusions: Less myocardial perfusion abnormalities during exercise than during adenosine stress in patients treated with beta-blockers may indicate less ischemia but also an impaired diagnostic performance. Thus adenosine stress test should be preferred to optimize the diagnostic sensitivity in patients during beta-blocker treatment.