Recruitment of Oct-1 protein to the octamer sequence of U6 promoter is critical for optimal transcription by RNA polymerase III. Here we report that p38 kinase inhibitors, SB202190 and SB203580, stimulated U6 promoter activity and this stimulation can be observed only in the presence of octamer sequence. SB202190-treated cell nuclear extract had about 50% increase in Oct-1 binding activity suggesting that the increased U6 promoter activity by p38 kinase inhibitor is mediated through Oct-1. Mutation in octamer sequence significantly reduced the SB202190-stimulated U6 promoter transcription and the distance between octamer and proximal sequence element of U6 promoter is also critical for the p38 kinase inhibitor-stimulated activity. Exogenous Oct-1 expression showed a concentration-dependent activation of U6 promoter that was further stimulated by the p38 kinase inhibitors. When cells were treated with p38 kinase inducer, hydrogen peroxide or phorbol 12-myristate 13-acetate (PMA), U6 promoter activity was down regulated and this inhibition was reversed by p38 kinase inhibitors. Over-expression of p38α kinase down-regulated U6 promoter activity and this inhibition was further enhanced by PMA and p38 kinase inhibitors reversed this inhibition. p38 kinase inhibitor-treated cells had 50% more U6 RNA than the control cells. Taken together, our results show a negative correlation between the p38 kinase levels and Oct-1 binding on U6 promoter, suggesting that U6 promoter is negatively regulated by p38 kinase.