The immunoglobulin kappa light chain locus has two enhancer elements: the intronic enhancer, which lies between the Jkappa cluster and the Ckappa exon, and the 3'kappa enhancer, which is located downstream of Ckappa. To address the contribution of these elements to the developmentally regulated activation of germline kappa locus transcription and rearrangement, we purified primary pro- and pre-B cells and determined by in vivo footprinting the sites within each enhancer that were occupied. We found that the kappa intronic enhancer NF-kappaB site is occupied in both pro- and pre-B cells, while CRE, BSAP, and PU.1/pip sites within the 3'kappa enhancer undergo changes in occupancy as cells progress from the pro-B to the pre-B cell stage. These findings suggest that regulation of the kappa locus in primary pre-B cells may be mediated by factors that bind the 3'kappa enhancer.