HIV-1 Vpr is a virion associated accessory protein that has multiple activities within an infected cell. One of the most dramatic effects of Vpr is the induction of cell cycle arrest at the G2/M boundary, followed by apoptosis. This effect has implications for CD4+ cell loss in AIDS. In normal cell cycle regulation, Wee1, a key regulator for G2-M progression, phophorylates Tyr15 on Cdc2 and thereby blocks the progression of cells into M. We demonstrate that Vpr physically interacts with Wee1 at the N-lobe of the kinase domain analogous to that present in other kinases. This interaction with Vpr enhances Wee1 kinase activity for Cdc2. Overexpression of Wee1 kinase-deficient mutants competes for Vpr mediated cell cycle arrest, and deletion of the region of Wee1 that binds Vpr abrogates that competition. However, the Vpr mutants I74P and I81P, which fail to induce G2 arrest, can bind to and increase the kinase activity of Wee 1 to the same extent as wild type Vpr. Therefore, we conclude that binding of Vpr to Wee 1 is not sufficient for Vpr to activate the G2 checkpoint, and it may, possibly, reflect an independent function of Vpr.