The objective of this study was to determine whether inducible nitric oxide (NO) synthase from a rat alveolar macrophage cell line (NR8383) activated by LPS plus IFN-gamma could be regulated by NO, one of the two products of the enzymatic reaction. This study was based on previous observations in this laboratory that NO is a negative feedback modulator of constitutive NO synthase from rat cerebellum. NO synthase activity was determined by monitoring the formation of 3H-L-citrulline from 3H-L-arginine in the presence of added cofactors. NO synthase catalyzed the conversion of L-arginine to equimolar quantities of NO and L-citrulline. NO and S-nitrosothiols inhibited NO synthase activity and this effect was enhanced by superoxide dismutase and attenuated by oxyhemoglobin. Nitrite and nitrate, the oxidation products of NO, as well as L-citrulline, the amino acid end-product, produced no significant effects on NO synthase activity. The inhibitory effect of NO on NO synthase appeared to be partially reversible upon addition of oxyhemoglobin. The inhibitory effect of NO was mimicked by other heme ligands including carbon monoxide, cyanide, and manganese-protoporphyrin IX. These observations indicate that (1) enzyme-bound heme plays a mechanistic role in the catalytic conversion of L-arginine to NO plus L-citrulline; (2) NO may function as a negative feedback modulator of inducible NO synthase by interacting with enzyme-bound heme; and (3) negative feedback modulation by NO may represent a mechanism by which the potentially toxic L-arginine-NO pathway in activated alveolar macrophages is turned off.