Arsenic acts as a toxicant, a carcinogen, and an effective chemotherapeutic agent, but its mechanisms of action are unclear. We have previously shown that treatment of U937 cells with 5 microM sodium arsenite inhibits cell cycle progression through each cell cycle phase, including S phase. Cdc25A dual specificity phosphatase controls entry into and progression through S phase by dephosphorylating sites of inhibitory phosphorylation on cyclin E-cdk2 (Thr14 and Tyr15). Immunoblotting reveals that a 3-h treatment of U937 cells with 5 microM sodium arsenite results in a dramatic decrease in cdc25A protein levels. Coimmunoprecipitation experiments confirm that cyclin E-cdk2 is more phosphorylated at Thr14 and Tyr15 in the presence of arsenite, and kinase activity assays reveal a decrease in cyclin E-associated cdk2 activity. Therefore, arsenite-dependent cdc25A depletion could contribute to S phase inhibition. There exists an S phase checkpoint known to be mediated by proteasomal cdc25A degradation. However, cycloheximide half-life assay reveals that cdc25A is actually stabilized in arsenite-treated cells. Real-time RT-PCR shows that cdc25A mRNA levels are substantially decreased with arsenite treatment, and actinomycin D half-life assay reveals no change in message stability. Decreased cdc25A message translation is shown by sucrose density gradient polysomal analysis to be an unlikely cause for the profound arsenite-dependent reduction in cdc25A protein levels. Studies are ongoing to establish the mechanism by which 5 microM arsenite decreases cdc25A message abundance, but we surmise that, given the lack of effect on mRNA stability, an inhibition of gene transcription is likely involved.