Viable, chronically dysfunctional myocardium with reduced resting flow (or hibernating myocardium) is an important prognostic factor in ischemic heart disease. Although ²⁰¹Thallium imaging is frequently used to assess myocardial viability in patients with ischemic cardiomyopathy, there is limited data regarding its deposition in hibernating myocardium, and this data suggests that Thallium retention may be super-normal as compared to control myocardium. Accordingly, pigs (n=7) were chronically-instrumented with a 1.5 mm Delrin stenosis on the proximal LAD to produce hibernating myocardium. Four months later, severe anteroapical hypokinesis was documented with contrast ventriculography (wall motion score 0.7±0.8, normal=3), and microsphere measurements confirmed reduced resting flow (LAD subendocardium 0.78±0.34 vs. 0.96±0.24 ml/min/g in Remote, p<0.001). Absolute deposition of ²⁰¹Thallium and insulin-stimulated [¹⁸F]-2 fluoro-2-deoxyglucose (FDG) were assessed over 1-hour and compared with resting flow (n=704 samples). ²⁰¹Thallium deposition was only weakly correlated with perfusion (r²=0.20, p<0.001), and was more homogeneously distributed (relative dispersion 0.12±0.03 vs. 0.29±0.10 for microsphere flow, p<0.01). Thus, after 1-hour relative ²⁰¹Thallium (subendocardium LAD/remote, 0.96±0.16) overestimated relative perfusion (0.78±0.32, p<0.0001), and underestimated the relative reduction in flow. Viability was confirmed by both histology and preserved FDG uptake. We conclude that under resting conditions, ²⁰¹Thallium redistribution in hibernating myocardium is nearly complete within 1-hour, with similar deposition to remote myocardium despite regional differences in flow. These data suggest that in this time-frame ²⁰¹Thallium deposition may not discriminate hibernating myocardium from dysfunction myocardium with normal resting flow. Since hibernating myocardium has been associated with a worse prognosis, this limitation could have significant clinical implications.