BOB.1/OBF.1 is a lymphocyte-restricted transcriptional coactivator. It binds together with the Oct1 and Oct2 transcription factors to DNA and enhances their transactivation potential. Mice deficient for the transcriptional coactivator BOB.1/OBF.1 show several defects in differentiation, function and signaling of B cells. In search of BOB.1/OBF.1 regulated genes we identified Btk--a cytoplasmic tyrosine kinase-as a direct target of BOB.1/OBF.1. Analyses of the human as well as murine Btk promoters revealed a non-consensus octamer site close to the start site of transcription. Here we show that Oct proteins together with BOB.1/OBF.1 are able to form ternary complexes on these sites in vitro and in vivo. This in turn leads to the induction of Btk promoter activity in synergism with the transcription factor PU.1. Btk, like BOB.1/OBF.1, plays a critical role in B cell development and B cell receptor signalling. Therefore the down-regulation of Btk expression in BOB.1/OBF.1-deficient B cells could be related to the functional and developmental defects observed in BOB.1/OBF.1-deficient mice.