Repeated sequence elements found upstream of the ribosomal gene promoter in Xenopus function as RNA polymerase I-specific transcriptional enhancers. Here we describe an in vitro system in which these enhancers function in many respects as in vivo. The principal requirement for enhancer function in vitro is the presence of a high concentration of upstream binding factor (UBF). This system is utilized to demonstrate that enhancers function by increasing the probability of a stable transcription complex forming on the adjacent promoter. Species differences in UBF are utilized to demonstrate that enhancers do not act by recruiting UBF to the promoter, rather UBF performs its own distinct role at the enhancers. UBF function in enhancement differs from that at the promoter, as it is flexible with respect to both the species of UBF and the enhancer element employed. Additionally, we identify a potential role for the mammalian UBF splice variant, UBF2, in enhancer function. We demonstrate that the TATA box binding protein (TBP)-containing component of Xenopus RNA polymerase I transcription, Rib1, can interact with an enhancer-UBF complex. This suggests a model in which enhancers act by recruiting Rib1 to the promoter.