Recently, we have demonstrated the feasibility of imaging myocardial uptake of fluorine-18 fluorodeoxyglucose (FDG) with single-photon emission tomography (SPET) using a specially designed collimator. Thallium-201 was used to determine distribution of perfusion for comparison with FDG uptake. However, regional201Tl and FDG activities may be different, based on differences in tracer attenuation and the use of different collimators. To study the relation between tracer activities imaged with SPET, nine healthy individuals underwent resting201Tl SPET and FDG SPET during a hyperinsulinaemic euglycaemic clamp. The SPET data were analysed semiquantitatively, using circumferential profiles. Mean profiles of midventricular short-axis slices showed no significant difference between201Tl and FDG activity. Regional fluctuations were similar for FDG and201Tl; the highest tracer activities were observed in the lateral wall and the lowest activities in the septum. Finally, pooled data (n=1620 segmental activities) in nine individuals showed a linear correlation (P<0.0001) between201Tl and FDG activity: FDG=0.76201Tl + 0.23 (r=0.70). These data demonstrate the absence of major differences between regional201Tl and FDG activity in normal myocardium, suggesting that no separate201Tl and FDG SPET reference values are needed for comparison with patient studies.