Repeated exposure to bacterial endotoxin causes a diminished response by the host to further exposure. One important feature of this hyporesponsiveness is a reduced macrophage production of nitric oxide (NO) via the inducible nitric oxide synthase (iNOS) pathway. Using a murine macrophage model, we observed that hyporesponsiveness was accompanied by a decrease in the levels of NO release (measured as nitrite), iNOS protein and iNOS gene transcription. The expression of the putative lipopolysaccharide (LPS) receptor, CD14, was not altered. In vivo genomic footprinting showed that the same binding sites are occupied in the iNOS promoter and enhancer of desensitized macrophages and of LPS-responsive macrophages, yet the composition of NF-kappaB in the nuclei of these cells was found to be altered. The transcriptionally inactive homodimer p50-p50 represented the predominant binding activity in nuclei from LPS-pretreated cells before and after stimulation. Nuclei from cells which had not been pretreated but were stimulated contained more of the transcriptionally active p50-p65 heterodimer than their pretreated counterparts. Consistent with this, the cytosolic steady-state level of an inhibitor of NF-kappaB activity, I-kappaBalpha, was decreased in normal cells but not in pretreated cells. We propose that the presence of an overwhelming excess of transcriptionally inactive p50 homodimers on their kappaB sites in the iNOS control region in pretreated cells may block kappaB site binding by p50-p65, thereby reducing the activity of the protein complex governing iNOS transcription.