The P4 promoter of parvovirus minute virus of mice (MVMp) directs transcription of the genes coding for nonstructural proteins. The activity of promoter P4 is regulated by several cis-acting DNA elements. Among these, a promoter-proximal GC box was shown to be essential for P4 activity (J.K. Ahn, B.J. Gavin, G. Kumar, and D.C. Ward, J. Virol. 63:5425-5439, 1989). In this study, a motif homologous to an Ets transcription factor-binding site (EBS), located immediately upstream from the GC box, was found to be required for the full activity of promoter P4 in the ras-transformed rat fibroblast cell line FREJ4. In normal parental FR3T3 cells, the transcriptional function of P4 EBS was insignificant but could be restored by transient cell transfection with the c-Ha-ras oncogene. P4 EBS may thus contribute to the stimulation of promoter P4 in ras-transformed cells. Electrophoretic mobility shift assays using crude extracts from FREJ4 cells revealed the binding of a member(s) of the Ets family of transcription factors to the P4 EBS, as well as the interaction of two members of the Sp1 family, Sp1 and Sp3, with the adjacent GC box. When produced in Drosophila melanogaster SL2 cells, Ets-1 and Sp1 proteins acted synergistically to transactivate promoter P4 through their respective cognate sites.