Objectives. We prospectively compared myocardial uptake of thallium-201 (Tl-201) at rest viith rest technetium-99m (Tc-99m) sestamibi uptake in the same patients, using quantitative single-photon emission computed tomography (SPECT). Background. Because of only slightly delayed redistribution, Tc-99m-sestamibi uptake at rest may be less than Tl-201 uptake, thereby underestimating the extent of viability. Methods. Twenty patients (2.25 stenoses per patient) with a mean left ventricular ejection fraction of 33 +/- 2% underwent early and 3-h delayed rest Tl-201 SPECT, rest Tc-99m-sestamibi SPECT and two-dimensional echocardiography. Results. The 280 scan segments were classified as either a normal, mild reduction in viability, defined as delayed Tl-201 uptake less than or equal to 75% and greater than or equal to 5%, or a severe reduction in viability, defined as delayed Tl-201 uptake <50%. Mild and severe defects were further classified as fixed or having rest Tl-201 redistribution, Comparisons by patients were made using repeated measures analysis of variance and Dunnett's multiple comparisons test to compare Tc-99m-sestamibi with initial rest Tl-201 and delayed Tl-201 uptake, Twenty patients had at least one mild fixed defect (95 total segments), The average percent uptake in these defects for initial Tl-201, delayed Tl-201 and Tc-99m-sestamibi was 62.5 +/- 2.7%, 63.1 +/- 7.1% and 67.3 +/- 9.7%, respectively (p = NS). Twelve patients (27 segments) had mild redistribution defects on serial rest Tl-201 imaging. The average percent uptake was 61.6 +/- 5.2% for initial Tl-201, 67.0 +/- 9.1% for delayed Tl-201 and 67.7 +/- 12.4% for Tc-99m-sestamibi defects, Technetium-99m sestamibi uptake was not significantly different than that for delayed Tl-201 but was significantly greater than initial Tl-201 uptake, Seventeen patients (52 segments) had severe fixed Tl-201 defects. The average percent uptake was 38.9 +/- 7.3% for initial Tl-201, 38.3 +/- 12.2% for delayed Tl-201 and 42.7 +/- 14.2% for Tc-99m-sestamibi defects in these patients (p = NS). Ten patients (19 segments) had severe redistribution defects on rest Tl-201 imaging, The average percent uptake was 37.0 +/- 8.5% for initial Tl-201, 42.9 +/- 8.6% for delayed Tl-201 and 44.5 +/- 11.3% for Tc-99m-sestamibi defects. As was seen for mild Tl-201 redistribution defects, Tc-99m-sestamibi uptake was significantly higher than initial Tl-201 uptake, but not significantly different than delayed Tl-201 uptake in these severe defects. Conclusions. Technetium-99m sestamibi uptake after injection at rest is comparable to Tl-201 uptake after injection at rest in patients with severe coronary artery disease and left ventricular dysfunction, suggesting comparable worth for viability assessment.