The aim of this study was to assess the influence of variations in the size of regions of interest (ROIs) on uptake values in brain tumours of L-3-iodine-123-alpha-methyl tyrosine (IMT). In addition, we attempted to establish the influence of size of ROIs on levels of significance assessing differences in mean IMT uptake between high-grade and low-grade tumours. Relative IMT uptake was determined in 19 patients with brain tumours using a MULTISPECT 3 triple-headed camera. Reconstructed image resolution was 14 mm at FWHM. Ten of the subjects suffered from high-grade gliomas (WHO grade III or IV) and nine from benign brain tumours, including eight patients with low-grade gliomas (WHO grade II). ROIs were defined by selecting those pixels within the tumour that exhibited uptake values above predefined threshold values. Using threshold values of 100, 95, 90, 85 and 80% of the mean, transaxial ROI size was approximately 0.1, 2.8, 4.3, 6.2 and 8.8 cm2, respectively. Over this range, mean, IMT uptake values decreased significantly from 2.4 to 1.9. High-grade tumours exhibited significantly higher IMT uptake than low-grade tumours at each of the threshold values. The corresponding levels of significance calculated using the Mann-Whitney U-test were between 0.01 and 0.02. Although IMT uptake values in brain tumours are significantly dependent on ROI size, levels of significance assessing differences in IMT uptake between high-grade and low-grade tumours are relatively insensitive to variations in this parameter.