Abstract Until recently, exposure of the hippocampus to prolonged elevated glucocorticoid levels was thought to result in damage and loss of pyramidal neurons. Most of the earlier studies were based on measures of neuronal density and used assumptions-based counting methods. Using a stereological technique, the optical fractionator, which eliminates potential biases inherent in the assumption-based techniques, we were able to demonstrate that chronic psychosocial stress in tree shrews has no effect on neuronal number in the hippocampal CA1 and CA3 regions. The present report will focus on the practical aspects of the optical fractionator, by describing in detail how to estimate the total number of neurons in the hippocampal CA1 and CA3 regions of tree shrews. In this example the group sizes have been increased over those used in the earlier study. The present study supports our previous conclusion that stress does not affect the number of hippocampal neurons in the CA1 and CA3 areas as suggested by other authors. The results obtained with the optical fractionator can be used to estimate the precision of the data.