L-arginine-dependent production of reactive nitrogen intermediates (RNIs: nitric oxide, nitrite, and nitrate) by mammalian macrophages has been proposed to occur via an L-arginine oxidative deimination pathway and is known to be responsible for certain antineoplastic and antimicrobial effector functions. The present study represents the first examination of this pathway in a non-mammalian vertebrate. Because chickens, unlike mammals, lack a urea cycle and are incapable of de novo synthesis of L-arginine, the possible existence of an avian macrophage pathway for production of RNIs is questionable. We have defined conditions under which chicken macrophages are able to produce nitrite. Sephadex-elicited chicken peritoneal macrophages required a bacterial lipopolysaccharide (LPS from Escherichia coli) signal to produce nitrite during 24 hour cultures in the presence of L-arginine. As little as 5 ng/ml LPS resulted in significant nitrite production in culture. The relationship of nitrite production to both LPS and L-arginine levels was dose-dependent. D-arginine was unable to substitute for L-arginine but also produced no inhibitory effect. In contrast, L-NG-monomethyl arginine showed a significant inhibitory effect on nitrite production. A virus-transformed chicken macrophage cell line, HD11, also produced nitrite in a dose-dependent manner relative to both LPS and L-arginine concentration. Concentrations as low as 5 ng/ml LPS and 0.1 mM L-arginine resulted in significant nitrite production, while maximum levels of nitrite production were obtained using greater than or equal to 0.5 micrograms/ml LPS and greater than or equal to 0.4 mM L-arginine. These results indicate that chicken macrophages can produce RNIs. This production is dependent upon activation and is influenced by local L-arginine concentration. Moreover, because the chicken does not possess the ability to synthesize arginine and has an absolute nutritional requirement for this amino acid, the chicken represents a highly controllable system to examine the in vivo effects of L-arginine on macrophage-related immune functions.