Recent studies from our laboratory have demonstrated that ascorbate potentiated enzymatic synthesis of nitric oxide (NO) from polymorphonuclear leukocytes (PMNs). NO is known to modulate various function of PMNs such as chemotaxis, adherence, aggregation, and generation of reactive oxygen species (ROS). The role of ascorbate in the PMN phagocytosis, ROS generation, and apoptosis was thus evaluated in the present study. Ascorbate and its oxidized and cell-permeable analog, dehydroascorbate (DHA), did not affect the phagocytosis but enhanced ROS generation and apoptosis following treatment with Escherichia coli or arachidonic acid. A detailed investigation on the DHA-mediated response indicated that inhibitors of DHA uptake, reduced nicotinamide adenine dinucleotide phosphate oxidase, NO synthase, or ROS scavengers attenuated ROS generation. In DHA-treated cells, enhanced generation of peroxynitrite was also observed; thus, ascorbate-mediated ROS and reactive nitrogen species generation might mediate cytotoxicity toward the ingested microbes and subsequently, augmented PMN apoptosis. Results of the present study have helped in delineating the role of ascorbate in the modulation of NO-mediated ROS generation from PMNs.