Abstract We have utilized the human 4F2 heavy chain (4F2HC) gene as a model system in studies designed to elucidate the molecular events involved in regulating inducible gene expression during normal human T-cell activation. In previous studies we have shown that steady state levels of 4F2HC mRNA are induced 50–60-fold within 6 h of T-cell activation by phytohemagluttinin (PHA) and that the induction of 4F2HC gene expression involves both the protein kinase C and calcium-mediated activation pathways. Despite the fact that the 4F2HC gene is highly regulated in T cells, the 5′ upstream region of the 4F2HC gene contains a housekeeping promoter which is G + C rich, lacks TATA or CCAAT sequences, and contains four potential binding sites for the ubiquitous Sp1 transcription factor. The major regulatory elements of the 4F2HC gene do not reside within this 5′ upstream region but instead, map to the exon 1-intron 1 region of the gene. The low levels of mature 4F2HC mRNA in resting T cells result from a block to transcription elongation within the exon 1-intron 1 region of the gene rather than promoter inactivity. Phorbol ester stimulation of resting T cells induces 4F2HC gene expression by removing this block to transcription elongation. We now report that in addition to its ability to serve as a transcriptional attenuator, the 4F2HC first intron contains a powerful enhancer element which is active in a wide variety of cell types including malignant human T cells. Full enhancer activity is displayed by a 186 bp fragment of the first intron which contains binding sites for two novel nuclear proteins (NF-4FA and NF-4FB) which flank a consensus binding site for the AP-1 transcription factor. A cDNA encoding the NF-4FB enhancer binding protein has been cloned by screening a lambda gt11 cDNA library with a rabiolabelled oligonucleotide corresponding to the NF-4FB recognition sequence.