There are currently four common types of stress used with thallium-201 imaging in the diagnosis of coronary artery disease and risk assessment. The objective of this study was to examine the thallium biokinetics during exercise, adenosine, dipyridamole and dobutamine stress testing in 15 healthy volunteers. Methods: Each subject underwent planar 201Tl imaging during maximal treadmill exercise testing, adenosine infusion (140 μg/kg/min for 6 min), dipyridamole infusion (142 μg/kg/min for 4 min) and dobutamine infusion (40 μg/kg/min). Results: Absolute myocardial thallium activity was greater after pharmacologic testing than exercise, (p < 0.001 each). Thus, the activity was 505 counts/pixel with adenosine, 491 counts/pixel with dipyridamole, 517 counts/pixel with dobutamine and 409 counts/pixel with exercise. The myocardial thallium clearance was lower with pharmacologic testing than exercise; 9.7%/hr with adenosine, 9.9%/hr with dipyridamole, 11.3%/hr with dobutamine and 13%/hr with exercise (p < 0.01 each). The thallium uptake and clearance in the lung and liver were also greater with pharmacologic stress testing than exercise (p < 0.05). Conclusions: Thus, thallium biokinetics are different during pharmacologic stress testing with adenosine, dipyridamole and dobutamine than during exercise. Diagnostic criteria for quantitative analysis of myocardial perfusion imaging must therefore be specific for the type of stress used.