We have shown that CD40 engagement induces TRAF1 gene expression in B lymphocytes. Here we report that CD40-dependent TRAF1 gene transcription in murine B cells is controlled by two enhancer regions. One region is located approximately 2 kb upstream of the transcription start site and the other lies in the intron between exons 5 and 6. The upstream enhancer contains a single NF-κB site in addition to sites that bind constitutive transcription factors. Mutation of this NF-κB site completely abrogates CD40-driven TRAFl transcription. The intronic enhancer contains two sites that strongly bind the CD40-inducible factors NF-κB and AP-1. Simultaneous mutation of the AP-1 site and of the NF-κB site abolishes transcription driven by this enhancer. When cloned together into reporter constructs, the two TRAF1 enhancers do not synergize, suggesting that each enhancer may separately participate in the induction of TRAF1 transcription in B cells following CD40 activation.