The POU domain transcription factor, Oct-2, is essential for the B cell-specific expression of CD36 in mouse B cells. In order to determine how Oct-2 mediates expression of CD36 in B cells, we cloned and analysed the mouse CD36 promoter. In contrast to the human CD36 promoter, the mouse promoter contains a consensus octamer element of the type ATGCTAAT. This octamer element can be bound by either Oct-1 or Oct-2 but requires the expression of Oct-2 to activate transcription in B cells. Mutation of the octamer element renders the CD36 promoter refractory to activation by Oct-2. Furthermore, we demonstrate that the CD36 octamer element does not support recruitment of the B cell-specific co-activator OBF-1 and that CD36 expression is unaffected in primary B cells derived from obf-1(-/-) mice. We conclude that Oct-2 activates CD36 gene expression in mouse B cells via the octamer element in the promoter. Our data also demonstrate that CD36 is the first example of an Oct-2-dependent gene whose expression in B cells is independent of OBF-1. These findings support the notion that Oct-2 regulates gene transcription by both OBF-1-dependent and -independent mechanisms.