Phosphorylation modulates the activity of many proteins that interact with nucleic acids including DNA and RNA polymerases. The HIV-1 reverse transcriptase (RT) is essential during the replicative cycle of the HIV-1 virus. HIV-1 RT has several potential sites for phosphorylation that could regulate its activities. In this work, the phosphorylation of HIV-1 RT is examined in vitro and in vivo, to evaluate any role for this modification in regulating RT metabolism. Recombinant unphosphorylated HIV-1 RT heterodimer expressed in bacteria can be phosphorylated in vitro by several purified mammalian protein kinases. Seven kinases were tested, and five of these enzymes phosphorylated HIV-1 RT. Using an insect baculovirus expression system, the 66 kDa HIV-1 RT was also phosphorylated in vivo. However, HIV-1 RT immunoprecipitated from H9-lymphoma cells infected with HIV-1 showed negligible phosphorylation. Our results indicate that purified HIV-1 RT can be phosphorylated by several mammalian protein kinases in vitro and during expression in baculovirus infected insect cells.