Purified vaccinia virus usually contains a large proportion of noninfectious virus which can be converted to infectious virus by incubation with purified plasma membrane. This activating reaction which is mediated by a heat stable component of the membrane has been studied. A suspension of liposomes containing the lipids extracted from plasma membrane of either KB cells or mouse RBCs activated the noninfectious virus in the same manner as heated plasma membrane. The phospholipid fraction of the KB cell lipids had the activating ability, but neither neutral lipid nor glycolipid fraction activated the virus. Liposomes containing phosphatidylserine activated the virus, whereas other tested phospholipids, including phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine, and sphingomyelin had no effect on virus infectivity. Lysolecithin reduced the infectivity. Treatment with isolated plasma membrane or liposomes increased hydrophobicity of the virus slightly, but did not change its density. Analysis of activated and then purified virus showed that all phospholipid species in the coincubated plasma membranes and liposome samples were transferred to the virus. The transfer was not a phospholipid exchange reaction but a one-way net transfer, and took place rapidly at 37 degrees to reach saturation within 1 hr of coincubation. Neither activation of virus nor transfer of phospholipid occurred when the mixture was incubated at a temperature below 8 degrees. The virus has great ability to extract phospholipids from coincubated lipid bilayer membranes, and association with phosphatidylserine gives the virus high infectivity.