Reverse transcriptases (RTs) alphabeta and beta from avian Rous sarcoma virus (RSV) harbor an integrase domain which is absent in nonavian retroviral RTs. RSV integrase contains a nuclear localization signal which enables the enzyme to enter the nucleus of the cell in order to perform integration of the proviral DNA into the host genome. In the present study we analyzed the subcellular localization of RSV RT, since previous results indicated that RSV finishes synthesis of the proviral DNA in the nucleus. Our results demonstrate that the heterodimeric RSV RT alphabeta and the beta subunit, when expressed independently, can be detected in the nucleus, whereas the separate alpha subunit lacking the integrase domain is prevalent in the cytoplasm. These data suggest an involvement of RSV RT in the transport of the preintegration complex into the nucleus. In addition, to analyze whether the integrase domain, located at the carboxyl terminus of beta, exhibits integration activities, we investigated the nicking and joining activities of heterodimeric RSV RT alphabeta with an oligodeoxynucleotide-based assay system and with a donor substrate containing the supF gene flanked by the viral long terminal repeats. Our data show that RSV RT alphabeta is able to perform the integration reaction in vitro; however, it does so with an estimated 30-fold lower efficiency than the free RSV integrase, indicating that RSV RT is not involved in integration in vivo. Integration with RSV RT alphabeta could be stimulated in the presence of human immunodeficiency virus type 1 nucleocapsid protein or HMG-I(Y).