Vimentin, a member of the intermediate filament protein family, is regulated both developmentally and tissue specifically. It is also a marker of the metastatic potential of many tumor cells. Pre viously, the human vimentin promoter has been shown to contain multiple elements for the binding of both positive- and negative-acting regulatory factors. Transient transfection analysis of various vimentin 5'-end promoter sequences and mutants thereof fused to a reporter gene further defined two regulatory elements, a positive element that binds Sp1 and a negative element that binds the protein ZBP-89. ZBP-89 has been shown to be either a repressor or an activator of gene expression, depending on the promoter. Here, we show that for vimentin, both ZBP-89 and ZBP-99 repress reporter gene expression in Schneider (S2) cells. Deletion constructs confirm that the glutamine-rich region of Sp1 is required to enhance vimentin transcription, whereas the N-terminus of ZBP-89 is required to interact with Sp1 and repress gene expression. The overexpression of hTAF(II)130 can alleviate ZBP-89 repression in S2 cells, suggesting how ZBP-89 might serve to block gene expression.