In this study, the downstream effects of thymidylate synthase (TS) inhibition in L1210 (p53 mutant) and HL60 (p53 null) leukaemia cells were investigated. TS inhibition was induced by the specific TS inhibitor Thymitaq. Within 24 h, TS inhibition resulted in S-phase cell cycle arrest in both cell lines and subsequent apoptotic cell death as characterized by nuclear condensation, DNA fragmentation and the formation of apoptotic bodies. A biphasic hyper/hypopolarization of the mitochondrial membrane potential (delta psi m) was also observed. The mitochondrial permeability transition inhibitor, cyclosporin A, increased the baseline level of delta psi m in L1210 cells. However, along with bongkrekic acid, it did not influence the changes in delta psi m induced by TS inhibition in either cell line. In both cell lines the broad spectrum caspase inhibitor, zVAD.fmk as a single agent, induced a significant downward shift in the baseline of delta psi m. However, only in HL60 cells was this accompanied by a slight increase in cytotoxicity. In L1210 cells zVAD.fmk inhibited DNA fragmentation induced by Thymitaq but did not influence other cell cycle events (S-phase arrest) or the biphasic mitochondrial alterations, indicating caspase involvement downstream but not upstream of the mitochondria following TS inhibition. In HL60 cells, zVAD.fmk reduced the hyperpolarization of delta psi m observed with Thymitaq alone and failed to inhibit the increase in the sub-G1 population induced by Thymitaq. Moreover, zVAD.fmk significantly increased the cell death response of these cells following TS inhibition. In conclusion, cell death induced by TS inhibition is mediated via the apoptotic pathway which clearly involves biphasic alterations in delta psi m. In L1210 cells, but not in HL60 cells, caspases function as the final executioner of apoptosis.