Exponentially growing cultures of human bladder tumor cells (RT4 and T24) were treated with Vitamin C (VC) alone, Vitamin K₃ (VK₃) alone, or with a VC:VK₃ combination continuously for 5 days or treated with vitamins for 1 h, washed with PBS and then incubated in culture medium for 5 days. Co-administration of the vitamins enhanced the antitumor activity 12- to 24-fold for the RT-4 cells and 6- to 41-fold for the T24 cells. Flow cytometry of RT4 cells exposed to the vitamins revealed a growth arrested population and a population undergoing cell death. Growth arrested cells were blocked near the G₀/G₁-S-phase interface, while cell death was due to autoschizis. Catalase treatment abrogated both cell cycle arrest and cell death which implicated hydrogen peroxide (H₂O₂) in these processes. The H₂O₂ production resulted in a moderate increase in lipid peroxidation and depletion of cell thiol levels. Analysis of cellular ATP levels revealed a transient increase in ATP production for VC and the VC:VK₃ combination, but decreased ATP levels following VK₃ treatment. Lipid peroxidation, thiol depletion and ATP modulation occurred at a 17-fold lower concentration in the vitamin combination than with either vitamin alone. These results suggested that the increased cytotoxicity of the vitamin combination was due to redox cycling and increased oxidative stress.