Q-Gene is an application for the processing of quantitative real-time RT-PCR data. It offers the user the possibility to freely choose between two principally different procedures to calculate normalized gene expressions as either means of Normalized Expressions or Mean Normalized Expressions. In this contribution it will be shown that the calculation of Mean Normalized Expressions has to be used for processing simplex PCR data, while multiplex PCR data should preferably be processed by calculating Normalized Expressions. The two procedures, which are currently in widespread use and regarded as more or less equivalent alternatives, should therefore specifically be applied according to the quantification procedure used. AVAILABILITY: Web access to this program is provided at http://www.biotechniques.com/softlib/qgene.html