Simultaneously acquired dual-isotope 201Tl/99mTc SPECT studies were performed using cardiac and thoracic phantoms to evaluate the dual-isotope myocardial perfusion technique. Cardiac phantom images representing infarction, viable myocardium and various levels of ischemia were analyzed. Studies with and without attenuating media were performed, and myocardium-to-defect count ratios and defect sizes from dual-isotope SPECT images were compared to myocardium-to-defect count ratios and defect sizes from single-isotope (201Tl and 99mTc) SPECT images. Dual-isotope studies also were interpreted qualitatively. Studies with background activity simulating clinical conditions were performed and interpreted qualitatively. Myocardium-to-defect count ratios from both 99mTc and 201Tl were similar in single-isotope and dual-isotope SPECT images. Thallium-201 and 99mTc defect sizes were decreased slightly (mean +/- s.d., 1.0 +/- 1.7 cc for 201Tl and 0.7 +/- 1.0 cc for 99mTc) on dual studies when compared to single studies but were not statistically significant. Dual-isotope image simulations of normal, ischemic and infarcted and viable myocardium were correctly identified by experienced clinicians in 95% of the cases (21/22). Simultaneous dual-isotope 201Tl/99mTc SPECT imaging of cardiac phantoms produced images that had similar myocardium-to-defect count ratios to those produced using single-isotope techniques and were correctly evaluated on qualitative analysis. Changes in defect size related to dual-isotope imaging were minimal and not qualitatively important.