Integration of viral DNA into a chromosome of the infected host cell is required for efficient replication of a retroviral genome, and this reaction is mediated by the virus-encoded enzyme integrase (IN). As IN plays a pivotal role in establishing infection during the early stages of the retroviral life cycle, it is an attractive target for therapeutic intervention. However, the lack of effective antiviral drug therapy against this enzyme has led to the testing of other novel approaches towards its inhibition. In these studies, a panel of anti-human immunodeficiency virus type 1 (anti-HIV-1) IN hybridomas has been used in the construction of single-chain variable antibody fragments (SFvs). The monoclonal antibodies produced by these hybridomas, and derived SFvs, bind to different domains within IN. We now demonstrate that intracellular expression of SFvs which bind to IN catalytic and carboxy-terminal domains results in resistance to productive HIV-1 infection. This inhibition of HIV-1 replication is observed with SFvs localized in either the cytoplasmic or nuclear compartment of the cell. The expression of anti-IN SFvs in human T-lymphocytic cells and peripheral blood mononuclear cells appears to specifically neutralize IN activity prior to integration and, thus, has an effect on the integration process itself. These data support our previous studies with an anti-HIV-1 reverse transcriptase SFv and demonstrate further that intracellularly expressed SFvs can gain access to viral proteins of the HIV-1 preintegration complex. This panel of anti-HIV-1 IN SFvs also provides the tools with which to dissect the molecular mechanism(s) directly involved in integration within HIV-1-infected cells.