Sampling of arterialised venous blood (AVB) is often used as an alternative to sampling of arterial blood when determining the myocardial metabolic rate of glucose (MRGlu). This method, however, has not yet been validated for measurement of plasma fluorine-18 fluorodeoxyglucose (FDG) activity during a euglycaemic-hyperinsulinaemic clamp (EHC). In this study, dynamic FDG scans were performed with arterial blood sampling during EHC. Samples of arterial and AVB or venous blood were simultaneously withdrawn at five time points for measurement of FDG activity and plasma glucose in 36 patients. Both venous to arterial and AVB to arterial ratios were calculated for FDG activity and plasma glucose. Mean ratios between AVB and arterial FDG activity were then used to create calculated arterialised venous input functions from corresponding arterial input functions. The mean effect of arterialisation on the calculation of K(i) was assessed. In nine additional patients, K(i) obtained with continuous sampling of AVB was compared with K(i) obtained with a corresponding (quality-controlled) image-derived input function from the ascending aorta. Using AVB, measurements of FDG activity were much more reliable than with venous blood sampling. As compared with arterial sampling, however, FDG activity was underestimated early after injection, while it was overestimated after 20 min. In both analyses, AVB resulted in approximately 10%+/-10% overestimation of K(i). Because of a 5%+/-5% underestimation of plasma glucose concentration with AVB, the net effect on the final calculation of MRGlu was small (on average 5% overestimation). It is concluded that the use of AVB has a small average effect on the determination of MRGlu. This method does, however, contribute to variability in the results. This variability cannot be explained by different degrees of arterialisation.