In cDNA microarray image processing, there are different methods for calculating the channel ratios. Standard microarray image analysis software, such as the Axon GenePix Pro, calculate the channel ratio from pixels that define a given spot using different methods (i.e. ratio of means, ratio of medians, mean of ratios, median of ratios, and regression ratio). Ratio values calculated using the different methods will then be listed in an output file. Microarray users have to choose one of the available methods at their own discretion, as no guidelines are provided. Therefore, we aim to address one of the most frequently asked questions by the microarray users: which ratio quantity provided by the image analysis software should be used? In this study, we have evaluated the five different ratio calculation approaches using simulation studies. Our results suggest that in most circumstances the ratio of means appears to be the best approach, particularly when the coefficient of variance (CV) of two-channel pixel intensities are small (<0.5) and channel intensities are large. Conversely, the ratio of medians and the median of ratios are more favourable when the CV is large.