Aims: Carcinogenesis is a multi-step process that is frequently associated with p53 inactivation. The class 1 carcinogen cadmium (Cd) causes renal cancer and is known to inactivate p53. G2/mitosis(M) arrest contributes to stabilization of p53-deficient mutated cells, but its role and regulation in Cd exposed p53-deficient renal cells is unknown. Methods: In p53-inactivated kidney proximal tubule cells (PTC), Comet assay was used for DNA damage, dihydrorhodamine 123 for formation of reactive oxygen species (ROS) and immunofluorescence microscopy with immunoblotting for phosphorylated proteins. Cell cycle phases were analysed by propidium iodide staining and flow cytometry. Cell viability was determined by MTT assay and H-33342 nuclear staining. Results: DNA damage was induced by Cd (50-100 mM) within 1-6 h. This was associated with peak ROS formation at 1-3 h and G2/M cell cycle arrest at 6 h, which were both abolished by the antioxidant a-tocopherol (100 mM). G2/M arrest by Cd was enhanced by ~2-fold upon release from cell synchronization (double thymidine block or nocodazole) and resulted in ~2-fold increase of apoptosis, indicating that G2/M arrest mirrors DNA damage and toxicity. The Chk1/2 kinase inhibitor UCN-01 (0.3 mM) relieved Cd-induced G2/M arrest and increased apoptosis. Furthermore, Cd increased phosphorylation of cyclin-dependent kinase cdc2 at tyrosine15, leading to inactivation and preventing entry of cells into mitosis, which was blocked by UCN-01. Conclusions: The data indicate that in p53-inactivated PTC Cd-induced ROS formation and DNA damage trigger signaling of check-point activating kinases ATM/ATR to cause G2/M arrest. This may promote survival of pre-malignant PTC and Cd carcinogenesis. Funded by DFG TH 345/10-1.