BOB.1/OBF.1 is a lymphocyte-restricted transcriptional coactivator. It binds to the Oct1 and Oct2 transcription factors and increases their transactivation potential. Targeted gene disruption experiments revealed that BOB.1/OBF.1 is critical at different stages of B cell development. A large number of genes expressed in B cells contain octamer motifs in their regulatory regions. However, only few genes have been described so far whose expression is dependent on BOB.1/OBF.1. To understand the molecular basis of BOB.1/OBF.1 function in B cell development, we searched for BOB.1/OBF.1 target genes by expression profiling. We have identified genes both induced and repressed by BOB.1/OBF.1. Using different genetic systems, we demonstrate regulation of a selection of these genes. Identified targets included genes encoding Ahd2-like, AKR1C13, Rbp1, Sdh, Idh2, protocadherin gamma, alpha-catenin, Ptprs, Id3, and Creg. Classification of BOB.1/OBF.1 target genes by function suggests that they affect various aspects of B cell physiology such as cellular metabolism, cell adhesion, and differentiation. To better understand the mechanism of BOB.1/OBF.1 action, we cloned the promoter of the gene encoding Ahd2-like, the gene showing the strongest regulation by BOB.1/OBF.1. This promoter indeed contains a perfect octamer motif. Furthermore, the motif was recognized by the Oct transcription factors as well as BOB.1/OBF.1 in vitro and in vivo, as shown by electromobility shift and chromatin immunoprecipitation assays. Transient transfections confirm that this promoter is activated by BOB.1/OBF.1. Our observations suggest that by regulating genes in different functional pathways, BOB.1/OBF.1 has a widespread effect on B cell development and function.