Biosynthesis of nitric oxide (.NO) from L-arginine by nitric oxide synthase (NOS) represents a major cytotoxic effector function of macrophages. It has been shown that most mammalian NOS requires tetrahydrobiopterin (BH4) as a cofactor and that inhibition of BH4 synthesis results in suppressed .NO production. Chicken L-arginine metabolism differs from that of mammals in that chickens cannot synthesize L-arginine de novo. Therefore, it is important to examine whether chicken macrophage .NO synthesis is also BH4-dependent. 2,4-diamino-6-hydroxypyrimidine (DAHP), a specific inhibitor for GTP cyclohydrolase I (GTP-CH; EC 3.5.4.16), the rate-limiting enzyme in de novo pterin synthesis, was used to block synthesis of BH4. Both chicken peritoneal macrophages (PECs) and the avian MC29 virus-transformed macrophage cell line, HD11, exhibited a dose-dependent reduction in .NO production (measured as nitrite accumulation) relative to DAHP concentration. Authentic BH4 and a substrate for pterin salvage pathway of BH4 synthesis, sepiapterin, were both capable of restoring the production of .NO in DAHP-treated PECs and HD11 macrophages. These results suggest that chicken macrophages require active synthesis of BH4 to produce .NO and that chemicals interfering with BH4 synthesis may result in suppressed .NO production and, hence, .NO-mediated immune function.