The dihydrofolate reductase (dhfr) promoter contains cis-acting elements for Sp1 and E2F. Here we examined the cooperative regulation of dhfr gene transcription by Sp1 and E2F in human osteosarcoma cells, U2OS. Trichostatin A, an inhibitor of histone deacetylases, markedly stimulated dhfr promoter activity, a response that was enhanced by the deletion of an E2F element. In contrast, deletion of the dhfr Sp1 binding sites completely abolished promoter stimulation by trichostatin A. Cotransfection assays showed that activation of dhfr transcription by expression of E2F1/DP1 requires the reiterated Sp1 elements, whereas activation by Sp1 was enhanced by the deletion of the E2F element. Expression of HDAC1 with Sp1 suppressed promoter activity and suppression was not alleviated by coexpression of E2F1/DP1. These results suggest that HDAC1 acts through Sp1 to repress dhfr promoter activity, and that the E2F element modulates the activity of Sp1 at the dhfr promoter through a cis-acting mechanism.