Cytokines induce the mouse macrophage cell line RAW 264.7 to express cytokine-inducible nitric oxide synthase (iNOS), which is active only in dimeric form. Because dimerization of purified iNOS subunits requires tetrahydrobiopterin, heme, and L-arginine, we investigated if availability of these factors also influences intracellular assembly of dimeric iNOS. Following exposure to cytokines, iNOS protein was found to accumulate in a near linear manner over 16 h of further culture. In contrast, dimeric iNOS accumulated at a slower rate that continuously decreased during culture, resulting in only 25% of the accumulated iNOS protein being in dimeric form by 16 h. Further experiments argued against dimer instability or L-arginine and tetrahydrobiopterin availability as factors limiting iNOS dimer accumulation. Blocking cellular NO synthesis with Nomega-nitro-L-arginine methyl ester (L-NAME) greatly increased iNOS dimer assembly, indicating NO synthesis limited iNOS dimerization. NO synthesis was found to prevent an increase in soluble heme level that was associated with iNOS induction in Nomega-nitro-L-arginine methyl ester-treated cells and also diminished heme insertion into iNOS. These NO-related defects were not reversed by adding heme precursors or hemin to the activated cell cultures. Measurement of iron release from activated cells demonstrated that endogenous NO synthesis substantially increased the release of 59Fe to the medium. These observations suggest that iNOS dimerization is limited to a large extent by iNOS NO synthesis. NO appears to limit intracellular assembly of dimeric iNOS by preventing heme insertion and decreasing heme availability.