Transcription of the gene encoding the transcriptional coactivator Oct-binding factor 1 (OBF-1)/OCA-B/Bob.1 is largely restricted to B cells. During B cell development OBF-1 expression shows two peaks, one in immature B cells in the bone marrow and the other in germinal center B cells. Promoter analysis has identified a cAMP response element (CRE)-binding site present in the OBF-1 proximal promoter that is crucial for activity in B cells and for the induction of OBF-1 expression upon stimulation with CD40 ligand/IL-4. Here we address the question of how transcription of the OBF-1 gene is restricted to B cells. Surprisingly, in transient transfection assays the OBF-1 proximal promoter exhibited an equally strong activity in B and non-B cells. In contrast, upstream promoter regions displayed B cell-specific properties, partly overlapping with DNaseI hypersensitive sites identified in this study. In mice, expression of a neomycin resistance gene under the control of a Polyoma enhancer/TK promoter cassette was restricted to B cells when integrated into the OBF-1 locus, but was ubiquitous when integrated into two other loci, Oct-1 or the large subunit of RNA polymerase II.Therefore, lineage commitment of the OBF-1 gene is promoter independent and is achieved by regulating the entire locus in a B cell-specific manner.