Human immunodeficiency virus type 2 (HIV-2) gene expression is regulated by upstream promoter elements, including the peri-Ets (pets) site, which mediate enhancer stimulation following treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). We previously showed that the oncoprotein DEK binds to the pets site in a site-specific manner. In this report, we show that binding to the HIV-2 pets site is modulated by treatment of U937 monocytic cells with TPA, an activator of protein kinase C. TPA treatment resulted in a reduction in the levels of DEK and the formation of a faster migrating pets complex in gel shift assays. We show further that the actions of TPA on pets binding can be duplicated by phosphatase treatment of nuclear proteins and is blocked with okadaic acid, a protein phospatase-2A (PP2A) inhibitor. Finally, we demonstrate that ectopic expression of the catalytic domain of PP2A can activate the HIV-2 enhancer/promoter alone or in synergy with TPA, an effect mediated in part through the pets site. These results suggest that, through an interaction with the protein kinase C pathway, PP2A is strongly involved in regulating HIV-2 enhancer-mediated transcription. This is a consequence of its effects on DEK expression and binding to the pets site, as well as its effects on other promoter elements. These findings have implications not only for HIV-2 transcription but also for multiple cellular processes involving DEK or PP2A.