A modified in vitro transcription system has been used to study the function of the cloned bZIP transcription factor SPA and the binding activity ESBF I in activating transcription from the bifactorial endosperm box region of the wheat prolamin LMWG-1D1 gene. Recombinant SPA expressed in Escherichia coli activated transcription from the endosperm box motif, and this was dependent upon the binding of the nuclear protein ESBF I. ESBF I did not activate transcription independently, but potentiated SPA-mediated transcriptional activation. ESBF I is likely to be the equivalent of, or contain the recently characterised DOF class of, Zn-finger protein called WPBF. These data provide new information about the interplay of members of the bZIP and DOF transcription factor families in regulating expression from bifactorial sites found in a variety of plant promoters.