Previously, we demonstrated that pyrrolidine dithiocarbamate (PDTC) induced G1 cell cycle arrest in vascular smooth muscle cells (VSMC) through inducing p21WAF1 expression. It has recently been reported that the transcription factors involved in p21WAF1 activation by certain signaling factors may vary in different cell types. However, little is known concerning the molecular mechanisms by which PDTC induces p21WAF1 gene expression in VSMC. In this report, we demonstrate that PDTC induces the p21WAF1 expression at the mRNA level. This increase in p21WAF1 gene expression was due to p38MAPK-dependent activation of the p21WAF1 promoter by PDTC. Transcription factor Sp1 binding site was identified as the cis-element for the activation of p21WAF1 promoter by PDTC, as determined by deletion and mutation analysis. In addition, gel shift and supershift assays demonstrated that this PDTC-responsive element binds specifically to the transcription factor Sp1. Treatment with SB203580, an inhibitor of the p38MAPK, significantly down-regulated transactivation of PDTC-induced Sp1. Finally, the transient expression of VSMC with dominant negative p38MAPK plasmid suppressed PDTC-stimulated Sp1 activity. In conclusion, we report the novel finding that transcription factor Sp1 that is involved in the p38MAPK-mediated control of p21WAF1 regulation on VSMC in response to PDTC has now been identified.