It has recently been reported that the transcription factors involved in p21WAF1 activation by certain signaling factors may vary in different cell types. However, the role and importance of the signaling pathway in the transcriptional regulation of p21WAF1 on vascular smooth muscle cells (VSMC) in response to benzyl isothiocyanate (BITC) has been unclear. In this report, we demonstrate that BITC induces the p21WAF1 expression at the transcriptional level. This increase in p21WAF1 gene expression was due to p38MAPK-dependent activation of the p21WAF1 promoter by BITC. Transcription factor Sp1 binding site was identified as the cis-element for the activation of p21WAF1 promoter by BITC, as determined by deletion and mutation analysis. In addition, gel shift and supershift assays demonstrated that this BITC-responsive element binds specifically to the transcription factor Sp1. Treatment with SB203580, an inhibitor of the p38MAPK, significantly downregulated transactivation of BITC-induced Sp1. Finally, the transient expression of VSMC with dominant negative p38MAPK plasmid suppressed BITC-stimulated Sp1 activity. In conclusion, we report that the transcription factor Sp1 involved in the p38MAPK-mediated control of p21WAF1 regulation on VSMC in response to BITC has now been identified.