Computer cell counting experiments were performed in order to examine the consequences of over- or under-counting. The three-dimensional reaggregate culture laboratory environment for cell counting was used as a model for computer simulation. The laboratory environment for aggregate and cell sizes, numbers and spatial placement in gelatin blocks was mimicked in the computer setup. However, in the computer, cell counting was set to be either ideally unbiased, or deliberately biased in regard to over- or under-counting so as to compare eventual results when using the various cell counting methods. It was found that there was no effect of the cell counting methods used in determining whether there was a significant difference in cell number between two experimental groups. In addition, it was found that under the conditions of these simulations, the optical disector method behaved similarly, on the average, as the ideal method of counting cell centers and in both of those cases, the average ratio between actual cell number in a flask and estimated number was close to 1.00. © 2001 Elsevier Science B.V. All rights reserved.