Human immunodeficiency virus type 1 (HIV-1) Vpr induces cell cycle arrest at the G2/M transition and subsequently apoptosis. Here we examined the potential involvement of Wee-1 in Vpr-induced G2 arrest. Wee-1 is a cellular protein kinase that inhibits Cdc2 activity, thereby preventing cells from proceeding through mitosis. We previously showed that the levels of Wee-1 correlate with Vpr-mediated apoptosis. Here, we demonstrate that Vpr-induced G2 arrest correlated with delayed degradation of Wee-1 at G 2/M. Experimental depletion of Wee-1 by a small interfering RNA directed to wee-1 mRNA alleviated Vpr-induced G2 arrest and allowed apparently normal progression through M into G1. Similar results were observed when cells were arrested at G2 following gamma irradiation. Thus, Wee-1 is integrally involved as a key cellular regulatory protein in the signal transduction pathway for HIV-1 Vpr-induced cell cycle arrest.