The expression of vimentin is unique within the intermediate filament multigene family. It is the only member which deviates from its usual tissue-specific expression pattern and whose 5'-flanking region contains multiple GC boxes, the binding site for Sp1. The activity of vimentin 5'-end:CAT fusions has been compared in cells where vimentin is highly expressed (mouse L cells) or not expressed at all (MH1C1). In addition, CAT activity has been examined by microinjection into Xenopus oocytes. Both in vivo expression and in vitro binding studies implicate Sp1 as a general regulatory factor in vimentin gene expression. Increased expression of 5'-end:CAT fusions in mouse L cells suggests that a fibroblast-specific enhancer element resides in the region -321 to -160. Low transcriptional activity in MH1C1 cells may be due to either the lack of this positive transcription factor(s) or the presence of a repressor element. Here, we demonstrate that the unique and complex pattern of vimentin gene expression is controlled by multiple cis-acting elements.