Both the hippocampus and amygdala are frequently targeted by researchers and clinicians for volumetric analysis based on magnetic resonance imaging (MRI). However, different data acquisition, analysis software and anatomical boundaries have in the past made it difficult to compare results of MRI studies from different laboratories. A reliable original segmentation protocol for volumetry of the amygdala and hippocampus on three-dimensional MRI was established by including merits of previous volumetric studies. Two skilled raters measured randomly selected 20 subjects from 242 subjects recently scanned in our institution (mean age 47.6 +/- 6.1 years). Data acquisition was performed with a three-dimensional spoiled gradient echo technique. Volumetric analysis was performed manually using three-dimensional software that allowed coronal, sagittal, and transverse images at any time. Both intra- and inter-rater reliability (intraclass correlation coefficients) yielded 0.99 for the hippocampus and 0.98 for the amygdala. It was confirmed that this segmentation protocol was highly reliable.