Simultaneous assessment of myocardial perfusion and function by gated single-photon emission tomography (GS) after a single tracer injection provides incremental information and is feasible with technetium-99m sestamibi. The present study validated the use of GS with thallium-201 for the assessment of left ventricular ejection fraction (LVEF) and regional wall motion by comparison with two-dimensional (2D) echocardiography (echo), which has not been done before. After injection of 111 MBq 201Tl at peak bicycle exercise (n = 55) or pharmacological stress (n = 17), GS was acquired 15 (post stress) and 120 min post injection (rest) on a double-head camera. An automatic algorithm (QGS) was used for processing. Echo (Acuson Sequoia C256) was performed immediately after rest GS. LVEFs assessed by GS and echo were correlated. The overall and segmental sensitivity and specificity of GS for the detection of regional wall motion abnormalities (WMAs) were calculated, echo serving as the gold standard. Perfusion abnormalities were scored. The success rate of the automatic algorithm was 100%, and visually assessed image quality was good to excellent in 88% of cases. Post-stress and rest LVEF as assessed by GS were highly correlated (r = 0.91). Good correlations were obtained between post-stress LVEF (GS) and rest LVEF (echo) and between rest LVEF (GS) and rest LVEF (echo) (r = 0.76 and 0.86 respectively). In patients with a reduced LVEF of less than 50% (n = 23), these correlations were even better (r = 0.84 and 0.89 respectively). Regional wall motion abnormalities (WMAs) were identified by GS with high sensitivity and specificity (88%-100% and 82%-98% respectively) and were directly related to the extent and severity of stress as well as of resting perfusion defects. It is concluded that GS with 201Tl is a feasible and reliable tool for the evaluation of patients with compromised left ventricular function in the context of coronary artery disease, and thus improves diagnosis and prognostic stratification. Regional WMAs were identified with high diagnostic accuracy and the method may prove helpful for the detection of myocardial viability.