Previous studies from this lab have demonstrated that in vitro ascorbate augments neutrophil nitric oxide (NO) generation and oxidative burst. The present study was therefore undertaken in guinea pigs to further assess the implication of ascorbate deficiency in vivo on neutrophil ascorbate and tetrahydrobiopterin content, NOS expression/activity, phagocytosis, and respiratory burst. Ascorbate deficiency significantly reduced ascorbate and tetrahydrobiopterin amounts, NOS expression/activity, and NO as well as free radical generation in neutrophils from scorbutics. Ascorbate and tetrahydrobiopterin supplementation in vitro, though, significantly enhanced NOS catalysis in neutrophil lysates and NO generation in live cells, but could not restore them to control levels. Although phagocytic activity remained unaffected, scorbutic neutrophils were compromised in free radical generation. Ascorbate-induced free radical generation was NO dependent and prevented by NOS and NADPH oxidase inhibitors. Augmentation of oxidative burst with dehydroascorbate (DHA) was counteracted in the presence of glucose (DHA uptake inhibitor) and iodoacetamide (glutaredoxin inhibitor), suggesting the importance of ascorbate recycling in neutrophils. Ascorbate uptake was, however, unaffected among scorbutic neutrophils. These observations thus convincingly demonstrate a novel role for ascorbate in augmenting both NOS expression and activity in vivo, thereby reinforcing oxidative microbicidal actions of neutrophils.