Retroviral integrase (IN) is expressed and incorporated into virions as part of the Gag-Pol polyprotein precursor. IN catalyzes integration of the proviral DNA into host cell chromosomes during the early stages of the virus life cycle, and as a component of Gag-Pol, it is involved in virion morphogenesis during late stages. It is unknown whether the scheme, conserved among retroviruses, for expressing and incorporating IN as a component of the Gag-Pol precursor protein is necessary for its function in the infected cell after viral entry. We have developed human immunodeficiency virus (H/V) virion-associated accessory proteins (Vpr and Vpx) as vehicles to deliver both foreign and viral proteins into the virus particle by their expression in trans as heterologous fusion proteins (X. Wu, et al., J. Virol. 69:3389- 3398, 1995; X. Wu, et al., J. Virol. 70:3378-3384, 1996; X. Wu, et al., EMBO J. 16:5113-5122, 1977). To analyze IN function independent of its expression as a part of Gag-Pol, we expressed and incorporated IN into HIV type 1 (HIV- 1) virions in trans as a fusion partner of Vpr (Vpr-IN). Our results demonstrate that the Vpr-IN fusion protein is efficiently incorporated into virions and then processed by the vital protease to liberate the IN protein. Virus derived from IN-minus provirus is noninfectious. However, this defect is overcome by trans complementation with the Vpr-IN fusion protein. Moreover, complemented virions are able to replicate through a complete cycle of infection, including formation of the provirus (integration). These results show, for the first time, that full IN function can be provided in trans, independent of its expression and incorporation into virions as a component of Gag-Pol. This finding also indicates that the IN domain of Gag- Pol is not required for the formation of infectious virions when IN is provided in trans. The ability to incorporate functional IN into retroviral particles in trans will provide unique opportunities to explore the function of this critical enzyme in a biologically relevant context, i.e., in infected cells as part of the nucleoprotein/preintegration complex.