By mutational analysis of the octamer-TATA box intervening region in the mouse SP6 kappa promoter, we have mapped two octamer-dependent, costimulatory regions, A and B. The A region was active in late B cells only, while the B region was active throughout B cell differentiation. The B region was TATA proximal and contained a heptamer and an E box of the E2A type that is common in Vkappa promoters. Mutation of the heptamer element did not decrease transcriptional stimulation from this region, but mutations in, or immediately 5' of, the E box core sequence did. A protein binding to this region could be detected in nuclear extracts. The complex could only partially be competed with a muE5 binding site and could not be supershifted with Abs raised to E2A gene products, indicating that it may represent a novel E-box binding complex. The A region was located proximal to the octamer and contained a CCCT element that is conserved both with regard to position and sequence in human VkappaII promoters. By mutational analysis, the transcriptional stimulatory activity was mapped to the CCCT element that also is part of an early B cell factor (EBF) binding site. In late B cells, a novel protein (FA), which did not bind to the EBF binding site in the mb1 promoter, interacted with the A region. This protein was found to be expressed at lower levels in early B cells as well as in HeLa cells. Thus, the octamer-flanking sequence contains positive control elements that may act independently but that differ in the stage of B cell differentiation at which they are active. One of these factors is an example of an ubiquitously expressed transcription factor that participate in differentiation-specific transcriptional activation.