Factors contributing to the quantification of the central benzodiazepine receptor binding potential in vivo using 123I-iomazenil and single photon emission computed tomography (SPECT) were analysed in phantom studies and in volunteers. SPECT was performed with the SME810 multidetector system. The Hoffman three-dimensional brain phantom was used to investigate linearity and reproducibility of SPECT results using different 123I activity concentrations. Dynamic and multislice SPECT scans were performed in nine healthy volunteers between 0 and 270 min after injection of 110 MBq 123I-iomazenil. Displacement studies were performed in three volunteers using 1 mg flumazenil administered intravenously (i.v.) at 250 min postinjection (p.i.). Regions of interest (ROIs) over the cerebral cortex and various subcortical nuclei were either fixed areas based on a stereotaxic brain atlas, or manually drawn, based on 60% isocontour lines. Interobserver variability of the ROI data and ratios derived from those data were estimated for two independent observers. The brain phantom measurements showed linearity with respect to 123I concentration and good reproducibility. The interobserver study showed a reasonable interobserver reliability for the large fixed ROIs. The displacement study showed about 50% displacement in all ROIs. Concentrations of 123I-iomazenil in the brain can be measured reliably with the SME810. Large fixed ROIs based on a stereotaxic atlas may be used reliably for quantification. The level of 4 cm above the cantomeatal line does not provide a useful reference region with a very low central benzodiazepine receptor binding potential.