The BOB.1/OBF.1/OCA-B protein (henceforth designated as BOB.1/OBF.1) is a B cellspecific co-activator of the Oct1 and Oct2 transcription factors. It is involved in mediating the transcriptional activity of these proteins. Surprisingly, animals deficient for BOB.1/OBF.1 showed normal expression of genes that contain an octamer motif in their regulatory regions. Here we have addressed the role of BOB.1/OBF.1 for octamer-dependent transcription. We show that promoters exclusively dependent on functional octamer motifs are completely inactive in BOB.1/OBF.1-deficient B cells. The lack of activity is a direct consequence of lack of the co-activator. To show this, a hormone-regulated conditional allele of BOB.1/OBF.1 was introduced into the BOB.1/OBF.1-deficient B cells. This resulted in the hormone-dependent transcriptional activity of octamer-dependent reporters in these cells. The BOB.1/OBF.1 requirement for octamer promoter function was also observed when an authentic immunoglobulin O -promoter was assayed. BOB.1/OBF.1 dependence could not be overcome by including the strong enhancer element from the immunoglobulin heavy chain gene. Induction of pre-B cells with lipopolysaccharide led to increased Oct2 levels but did not significantly increase octamer-dependent transcription in BOB.1/OBF.1-deficient B cells. Thus, these results demonstrate that BOB.1/OBF.1 itself is a non-redundant protein in B cells and absolutely required for octamer-dependent transcriptional activity.