The induction of immunoglobulin heavy chain (IgH) 3' enhancer activity has been coupled to ligand/receptor-dependent activation of resting B cells. To search for transcriptional target sites that account for this induction, extracts from lipopolysaccharide (LPS)-stimulated B cells and cell lines were used. Here we describe, by gel-retardation analysis, the identification of an NF-kappaB site and an adjacent nuclear factor ets-like (NFE) site in the 3' enhancer. The NFE motif binds four protein complexes in resting B cell extracts, of which two are down-regulated upon LPS stimulation. Gel shift-shift experiments of the NF-kappaB complexes with specific antibodies identified p50 and c-Rel proteins to be the predominant factors in primary LPS-stimulated cell extracts. Site-directed mutagenesis of these motifs demonstrates that they contribute to part of the enhancer activity in plasma cells. One copy of the NFkappaB/NFE motifs, linked to a heterologous reporter construct, displays lymphoid-restricted reporter gene activity in transient transfection assays. Mutation of either site abrogates all promoter activity. Complementation experiments demonstrate that although p50 and c-Rel expression vectors reconstitute transcription of an intact NF-kappaB/NFE reporter construct in a dose-dependent manner, mutation of the NFE site or the NF-kappaB site abrogates essentially all transcriptional activity in both plasma cells and in COS cells. Taken together, we provide evidence for the existence of an activator, NFE, which in combination with the p50 and c-Rel proteins, are part of the transcription factor machinery that regulates 3' enhancer activity, and thus the control of the IgH locus in late B lymphocyte development.