Human immunodeficiency virus type 1 (HIV-1) replication requires reverse transcription of its RNA genome into a double-stranded cDNA copy, which is then integrated into the host cell chromosome. The essential steps of reverse transcription and integration are catalyzed by the viral enzymes reverse transcriptase (RT) and integrase (IN), respectively. In vitro, HIV-1 RT can bind with IN, and the C-terminal domain (CTD) of IN is necessary and sufficient for this binding. To better define the RT-IN interaction, we performed nuclear magnetic resonance (NMR) spectroscopy experiments to map a binding surface on the IN CTD in the presence of RT prebound to a duplex DNA construct that mimics the primer-binding site in the HIV-1 genome. To determine the biological significance of the RT-IN interaction during viral replication, we used the NMR chemical shift mapping information as a guide to introduce single amino acid substitutions of nine different residues on the putative RT-binding surface in the IN CTD. We found that six viral clones bearing such IN substitutions (R231E, W243E, G247E, A248E, V250E, and I251E) were noninfectious. Further analyses of the replication-defective IN mutants indicated that the block in replication took place specifically during early reverse transcription. The recombinant INs purified from these mutants, though retaining enzymatic activities, had diminished ability to bind RT in a cosedimentation assay. The results indicate that the RT-IN interaction is functionally relevant during the reverse transcription step of the HIV-1 life cycle. OBJECTIVE To establish a productive infection, human immunodeficiency virus type 1 (HIV-1) needs to reverse transcribe its RNA genome to create a double-stranded DNA copy and then integrate this viral DNA genome into the chromosome of the host cell. These two essential steps are catalyzed by the HIV-1 enzymes reverse transcriptase (RT) and integrase (IN), respectively. We have shown previously that IN physically interacts with RT, but the importance of this interaction during HIV-1 replication has not been fully characterized. In this study, we have established the biological significance of the HIV-1 RT-IN interaction during the viral life cycle by demonstrating that altering the RT-binding surface on IN disrupts both reverse transcription and viral replication. These findings contribute to our understanding of the RT-IN binding mechanism, as well as indicate that the RT-IN interaction can be exploited as a new antiviral drug target.