Primary mixed liver cells were isolated from rats that had been fed an amino acid (AA) diet in which natural protein was replaced with a defined mixture of pure AAs. Nitric oxide (NO) production from these cells in vitro was monitored using a nitric oxide (NO)-selective fluorescent probe, diaminofluorescein, followed by flow cytometric analysis. High levels of NO fluorescence were seen in approximately half of liver cells isolated from rats fed an AA diet for 1-7 days, whereas there was baseline fluorescence in cells obtained from regular diet-fed rats. The apparent size of NO-producing cells was smaller than those not producing NO. The production of NO was inhibited when rats were treated with either inducible NO synthase (iNOS)- or endothelial NOS-specific inhibitor, and an inhibitor for iNOS induction during AA diet feeding. L-arginine or L-glutamine (material for L-arginine biosynthesis) enriched diet showed the same NO augmentation as in AA diet. It is speculated that a high content of free L-arginine in AA diet may have caused enhanced NO production.