PURPOSE: We investigated the intraobserver reproducibility of myocardial blood flow (MBF) measurements with PET at rest and during cold pressor test (CPT), and the interobserver agreement. METHODS: Twenty normal volunteers were studied. Using (13)N-ammonia, MBF was measured at rest and during CPT and measurement was repeated in a 1-day session (short-term reproducibility; SR). After a follow-up of 2 weeks, MBF was measured again at rest and during CPT and compared with the initial baseline measurement (long-term reproducibility; LR). In addition, adenosine-induced hyperemic MBF increases were assessed. RESULTS: Assessment of the SR did not show a significant absolute difference in MBF at rest, MBF during CPT or the endothelium-related change in MBF from rest to CPT (DeltaMBF) (0.09 +/- 0.10, 0.11 +/- 0.09, and 0.08 +/- 0.05 ml/g/min; p = NS), and they were linearly correlated (r = 0.72, r = 0.76 and r = 0.84; p < 0.0001). Corresponding values for standard error of the estimate (SEE), as indicative for the range of MBF measurement error, were 0.14, 0.14, and 0.09 ml/g/min. The LR yielded relatively higher but non-significant absolute differences in the MBF at rest, MBF during CPT and DeltaMBF (0.10 +/- 0.10, 0.14 +/- 0.10, and 0.19 +/- 0.10 ml/g/min; p = NS), and paired MBFs significantly correlated (r = 0.75, r = 0.71, and r = 0.60; p < 0.001). Corresponding SEEs were 0.13, 0.15, and 0.16 ml/g/min. The interobserver analysis yielded a high correlation for MBF at rest, MBF during CPT, and hyperemic MBF (r = 0.96, SEE=0.04; r = 0.78, SEE=0.11; and r = 0.87, SEE=0.28; p < 0.0001, respectively), and also a good interobserver correlation for DeltaMBF (r = 0.62, SEE=0.09; p < 0.003). CONCLUSION: Short- and long-term MBF responses to CPT, as an index for endothelium-related coronary vasomotion, can be measured reproducibly with (13)N-ammonia PET. In addition, the high interobserver reproducibility for repeat analysis of MBF values suggests the measurements to be largely operator independent.