Human immunodeficiency virus type 1 (HIV-1) Nef activation of p21-activated kinase 2 (PAK-2) was recapitulated in a cell-free system consisting of in vitro-transcribed RNA, rabbit reticulocyte lysate, and microsomal membranes on the basis of the following observations: (i) Nef associated with a kinase endogenous to the rabbit reticulocyte lysate that was identified as PAK-2, (ii) Nef-associated kinase activity was detected with Nefs from HIV-1(SF2), HIV-1(YU2), and SIV(mac239), (iii) kinase activation was not detected with a myristoylation-defective Nef (HIV-1(SF2)NefG2A) or with a Nef defective in PAK-2 activation but fully competent in other Nef functions (HIV-1(SF2)NefF195I), and (iv) Nef-associated kinase activation required activated endogenous p21 GTPases (Rac1 or Cdc42). The cell-free system was used to analyze the mechanism of Nef activation of PAK-2. First, studies suggest that the p21 GTPases may act transiently to enhance Nef activation of PAK-2 in vitro. Second, addition of wortmannin to the cell-free system demonstrated that Nef activation of PAK-2 does not require PI 3-kinase activity. Third, ultracentrifugation analysis revealed that whereas the majority of Nef and PAK-2 partitioned to the supernatant, Nef-associated PAK-2 activity partitioned to the membrane-containing pellet as a low-abundance complex. Lastly, Nef activation of PAK-2 in vitro requires addition of microsomal membranes either during or after translation of the Nef RNA. These results are consistent with a model in which activation of PAK-2 by Nef occurs by recruiting PAK-2 to membranes. As demonstrated herein, the cell-free system is a new and important tool in the investigation of the mechanism of PAK-2 activation by Nef.