The promoter of the human vimentin-encoding gene (VIM) contains two enhancers separated by a negative region. The distal and proximal enhancers bind the transcription factors, AP-1 and NK-kappaB, respectively, which contribute to serum induction of Vim synthesis. We were interested in looking for particular regulatory elements that might be responsible for tissue-specific extinction and culture-dependent activation of human VIM. We have identified a 48-bp sequence in the distal enhancer which had not been reported before. This sequence includes a negative element, NE2, which confers transcriptional repression in transfection experiments and binds at least two factors in vitro. NE2 may participate in the differentiation-stage-specific control of VIM expression which involves multiple regulatory sequences and several positive and negative trans-acting factors.