We have examined the integrity of J774 cell nitric oxide (NO) production and glutathione maintenance, whilst NADPH supply was compromised by inhibition of the pentose pathway with 6-aminonicotinamide. In resting cells 6-phosphogluconate accumulation began after 4 h and glutathione depletion after 24 h of 6-aminonicotinamide treatment. Cellular activation by lipopolysaccharide/interferon-lambda decreased glutathione by approximately 50% and synchronous 6-aminonicotinamide treatment exacerbated this to 31.2% of control (P < 0.05). In activated cells NO2- production was inhibited by 60% with 6-aminonicotinamide (P < 0.01), and superoxide production by 50% (P < 0.01) in zymosan-activated cells. NADPH production via the pentose pathway is therefore important to sustain macrophage NO production whilst maintaining protective levels of glutathione.