The role of two distinct kappa B sequence motifs found in the promotor of the murine IP-10 gene was studied in the transcriptional response of macrophages to lipopolysaccharides (LPS). When the murine macrophage cell line RAW 264.7 was stimulated with LPS, at least three different kappa B sequence-specific complex-forming activities were observed in nuclear extracts as assayed by electrophoretic mobility shift assay (EMSA). These three complexes were distinguished from one another in terms of time of appearance following stimulation and selectivity for one of the two different kappa B sequence motifs. The participation of individual members of the Rel homology family of kappa B sequence binding factors was assessed by use of specific antibodies in combination with either EMSA or UV-cross-linking to radiolabeled, BrdUrd-substituted oligonucleotide probes. The C1 complex contained predominantly NF kappa B1 (p50). The C2 complex contained NF kappa B1, RelA (p65), and perhaps other factors. The C3 complex contained predominantly c-Rel. Both kappa B sequences were able to mediate reporter gene transcription in LPS-stimulated macrophages, but the sites behaved differentially in cells co-transfected with expression vectors encoding different members of the Rel homology family. The results indicate that LPS activates several different forms of kappa B binding activity in murine macrophages which are composed of at least three different members of the Rel homology family. These binding activities exhibit differential recognition of and functional activation through the two distinct kappa B sequence motifs.