As part of the Fourth International Workshop on Genotoxicity Testing (IWGT), held 9-10 September 2005 in San Francisco, California, an expert working group on the Comet assay was convened to review and discuss some of the procedures and methods recommended in previous documents. Particular attention was directed at the in vivo rodent, alkaline (pH >13) version of the assay. The aim was to review those protocol areas which were unclear or which required more detail in order to produce a standardized protocol with maximum acceptability by international regulatory agencies. The areas covered were: number of dose levels required, cell isolation techniques, measures of cytotoxicity, scoring of comets (i.e., manually or by image analysis), and the need for historical negative/positive control data. It was decided that a single limit dose was not sufficient although the required number of dose levels was not stipulated. The method of isolating cells was thought not to have a qualitative effect on the assay but more data were needed before a conclusion could be drawn. Concurrent measures of cytotoxicity were required with histopathological examination of tissues for necrosis or apoptosis as the "Gold Standard". As for analysing the comets, the consensus was that image analysis was preferred but not required. Finally, the minimal number of studies required to generate a historical positive or negative control database was not defined; rather the emphasis was placed on demonstrating the stability of the negative/positive control data. It was also agreed that a minimum reporting standard would be developed which would be consistent with OECD in vivo genotoxicity test method guidelines.