The immunoglobulin kappa light chain intronic enhancer (iEkappa) activates kappa rearrangement and is required to maintain the earlier or more efficient rearrangement of kappa versus lambda (lambda). To understand the mechanism of how iEkappa regulates kappa rearrangement, we employed homologous recombination to mutate individual functional motifs within iE(kappa) in the endogenous kappa locus, including the NF-kappaB binding site (kappaB), as well as kappaE1, kappaE2, and kappaE3 E boxes. Analysis of the impacts of these mutations revealed that kappaE2 and to a lesser extent kappaE1, but not kappaE3, were important for activating kappa rearrangement. Surprisingly, mutation of the kappaB site had no apparent effect on kappa rearrangement. Comparable to the deletion of the entire iEkappa, simultaneous mutation of kappaE1 and kappaE2 reduces the efficiency of kappa rearrangement much more dramatically than either kappaE1 or kappaE2 mutation alone. Because E2A family proteins are the only known factors that bind to these E boxes, these findings provide unambiguous evidence that E2A is a key regulator of kappa rearrangement.