The Ames test is widely used in the screening of chemicals and compounds for potential carcinogenic effect. There is, however, considerable inter-laboratory variability in results from this assay. Using data from the RTI Collaborative Study of the EPA Ames Test Protocol, we show that their reported standard errors of estimates of mutagenicity fall far short of capturing day-to-day or laboratory-to-laboratory variation. We estimate the factors by which the standard errors must be inflated to account for these sources of variation. The laboratory protocol and previous studies suggest that much of this variation may be caused by factors that are relatively constant within days (e.g. technician, incubation temperature, S9 liver homogenate preparation) but vary over days and across laboratories. Therefore, such variation might be reduced through use of a reference compound tested on the same day and under the same conditions as the test chemical. This conjecture was, however, not supported by analyses that considered the positive control compound and a pure chemical as possible reference assays.