Recent findings have established caspase-2 as an important apical regulator in apoptotic pathways leading from DNA damage to release of mitochondrial cytochrome c and subsequent activation of effector caspases. Yet, the molecular map connecting the embarking stimuli of genotoxic stress with caspase-2 activation remains to be elucidated. Here, we address the question of potential caspase-2 regulators by examining 5-fluorouracil (5-FU)-induced apoptosis in wild-type and p53-deficient human colon carcinoma cells. Apoptosis was observed only in p53(+/+) cells and was preceded by caspase-2 activation. Hence, although no direct interaction between p53 and caspase-2 was observed in the cell system used, our data clearly demonstrate that a functional connection between these two proteins is essential for initiation of the 5-FU-induced apoptotic process. Proposed mediators of caspase-2 activation include PIDDosome complex proteins PIDD and RAIDD. Surprisingly, the presence of a complex encompassing at least RAIDD, PIDD and caspase-2 was verified in both p53(+/+) and p53(-/-) cells, also in the absence of 5-FU treatment. Thus, our results confirm the participation of PIDD and RAIDD in PIDDosome complex formation but question their role as sole mediators of caspase-2 activation. This assumption was further supported by siRNA transfections targeting PIDD or RAIDD. In conclusion, our findings support the hypothesis of p53 as an upstream regulator of caspase activity and provide data concerning caspase-2 processing mechanisms. As suppression of caspase-2 expression in 5-FU-treated cells also affects the level of the p53 protein, possibilities of a reciprocal interaction between these proteins are discussed.