Like all retroviruses, human immunodeficiency virus type 1 (HIV-1) undergoes reverse transcription during its replication cycle. The cellular cofactors potentially involved in this process still remain to be identified. We show here that A-kinase anchoring protein 149 (AKAP149) interacts with HIV-1 reverse transcriptase (RT) in both the yeast two-hybrid system and human cells. The AKAP149 binding site has been mapped to the RNase H domain of HIV-1 RT. AKAP149 silencing by RNA interference in HIV-1-infected cells inhibited viral replication at the reverse transcription step. We selected single-point mutants of RT defective for AKAP149 binding and demonstrated that mutant G462R, despite retaining significant intrinsic RT activity in vitro, failed to carry out HIV-1 reverse transcription correctly in infected cells. This suggests that the interaction between RT and AKAP149 in infected cells may play an important role in HIV-1 reverse transcription.