Immunosuppressive molecules within the aqueous humor (AqH) are thought to preserve ocular immune privilege by inhibiting pro-inflammatory nitric oxide (NO) production by macrophages (Mϕs). Consistent with previous observations, we observed that although Mϕs stimulated in the presence of AqH expressed NO-synthase-2 (NOS2) protein, nitrite concentrations in culture supernatants, an indirect measure NO production, did not increase. Interestingly, NOS2 enzymatic activity, as measured by the conversion of L-arginine (L-Arg) into L-citrulline, was augmented in lysates of Mϕs stimulated in the presence of AqH. These data suggested that intracellular L-arg may have been limited by AqH. However, we observed increased mRNA expression of the L-arg transporter, CAT2B, and increased L-arg uptake in Mϕs stimulated in the presence of AqH. Arginases were expressed by stimulated Mϕs but competition for L-arg with NOS2 was excluded. Expression of GTP cyclohydrolase which produces tetrahydrobiopterin (H4B), an essential cofactor for NOS2 homodimerization, increased after Mϕ stimulation in the presence or absence of AqH and NOS2 homodimers formed. Taken together these data provided no evidence for inhibited NOS2 enzymatic activity by AqH suggesting that a factor within AqH may have interfered with the measurement of nitrite. Indeed, we observed that nitrite standards were not measurable in the presence of AqH and this effect was due to ascorbate in AqH. Controlling for interference by ascorbate revealed that AqH augmented NO production in Mϕs via ascorbate which limited degradation of H4B. Therefore, AqH may augment NO production in macrophages by stabilizing H4B and increasing intracellular L-Arg.