The tissue- and stage-specific assembly of Ig and TCR genes is mediated by a common V(D)J recombinase complex in precursor lymphocytes. Directed alterations in the accessibility of V, D, and J gene segments target the recombinase to specific Ag receptor loci. Accessibility within a given locus is regulated by the functional interaction of transcription factors with cognate enhancer elements and correlates with the transcriptional activity of unrearranged gene segments. As demonstrated in our prior studies, rearrangement of the Igkappa locus is regulated by the inducible transcription factor NF-kappaB. In contrast to the Igkappa locus, known transcriptional control elements in the Iglambda locus lack functional NF-kappaB binding sites. Consistent with this observation, the expression of assembled Iglambda genes in mature B cells has been shown to be NF-kappaB independent. Nonetheless, we now show that specific repression of NF-kappaB inhibits germline transcription and recombination of Iglambda gene segments in precursor B cells. Molecular analyses indicate that the block in NF-kappaB impairs Iglambda rearrangement at the level of recombinase accessibility. In contrast, the activities of known Iglambda promoter and enhancer elements are unaffected in the same cellular background. These findings expand the range of NF-kappaB action in precursor B cells beyond Igkappa to include the control of recombinational accessibility at both L chain loci. Moreover, our results strongly suggest the existence of a novel Iglambda regulatory element that is either directly or indirectly activated by NF-kappaB during the early stages of B cell development.