Objectives. This study compares technetium-99m sestamibi (sestamibi) electrocardiographic (ECG) gated single-photon emission computed tomography (gated SPECT) and echocardiography for the evaluation of myocardial function and assesses the feasibility of single-injection, single-acquisition stress perfusion/rest function technetium-99m sestamibi-gated SPECT as an alternative to conventional stress/rest imaging for assessment of myocardial perfusion and viability. Background. Simultaneous assessment of stress perfusion and rest function is possible with gated SPECT acquisition of stress-injected technetium-99m sestamibi. Methods. Rest thallium-201 SPECT followed by stress sestamibigated SPECT (acquired 0.5 to 1 h after sestamibi injection) was performed in 58 patients. Echocardiography was performed immediately after or before gated SPECT in 43 of the patients. All studies were analyzed by semiquantitative visual scoring. Sestamibi-gated SPECT studies were read for stress perfusion and rest wall motion and thickening. Reversibility on sestamibi-gated SPECT was defined as the presence of a definite stress defect with normal or mildly impaired wall motion or thickening on gated SPECT. Results. There was high segmental score agreement between gated SPECT and echocardiography for wall motion (91%, kappa = 0.68, p < 0.001) and thickening (90%, kappa = 0.62, p < 0.001). Correlation for global wall motion (r = 0.9S, p < 0.001) and thickening (r = 0.96, p < 0.001) scores between the two modalities was excellent. In 32 patients without previous myocardial infarction, there was excellent agreement for reversibility between stress sestamibi-gated SPECT and rest thallium-201/stress sestamibi (98%, kappa = 0.93, p < 0.01). However, in 26 patients with previous infarction, discordance between the two approaches was frequent, with 26% (20 of 78) of nonreversible defects by stress sestamibi-gated SPECT being reversible by rest thallium-201/stress sestamibi and 21% (23 of 112) of reversible defects by stress sestamibi-gated SPECT being nonreversible by rest thallium-201/stress sestamibi. Conclusions. Gated SPECT of stress-injected sestamibi correlates well with echocardiographic assessment of regional function and thus adds information to perfusion SPECT. In patients without previous myocardial infarction, a single-injection stress perfusion/rest function approach using sestamibi-gated SPECT can substitute for conventional stress/rest myocardial perfusion imaging, adding a rest perfusion study only if there are nonreversible defects or consideration of attenuation artifacts. In patients with previous myocardial infarction, the gated SPECT approach does not replace the need for a rest perfusion study.