The alkaline single cell gel electrophoresis (comet) assay was used to assess in vitro and in vivo genotoxicity of etoposide, a topoisomerase II inhibitor known to induce DNA strand breaks, and chlorothalonil, a fungicide widely used in agriculture. For in vivo studies, rats were sacrificed at various times after treatment and the induction of DNA strand breaks was assessed in whole blood, bone marrow, thymus, liver, kidney cortex and in the distal part of the intestine. One hour after injection, etoposide induced DNA damage in all organs studied except kidney, especially in bone marrow, thymus (presence of HDC) and whole blood. As observed during in vitro comet assay on Chinese hamster ovary (CHO) cells, dose- and time-dependent DNA effects occurred in vivo with a complete disappearance of damage 24 h after administration. Even though apoptotic cells were detected in vitro 48 h after cell exposure to etoposide, such a result was not found in vivo. After chlorothalonil treatment, no DNA strand breaks were observed in rat organs whereas a clear dose-related DNA damage was observed in vitro. The discrepancy between in vivo and in vitro models could be explained by metabolic and mechanistic reasons. Our results show that the in vivo comet assay is able to detect the target organs of etoposide and suggest that chlorothalonil is devoid of appreciable in vivo genotoxic activity under the protocol used.