The replication cycle of HIV-1 and other retroviruses is characterized by reverse transcription of the viral RNA genome into a double-stranded DNA, which subsequently integrates into the host cell genome. This process is mediated by the virion-associated enzyme reverse transcriptase (RT), and the cellular tRNA ^lys3 molecule is used as a primer by HIV-1. In this review, we will discuss the mechanism of initiation of reverse transcription. The tRNA primer binds with its 3`-terminal 18 nucleotides to a complementary sequence in the viral genome, the primer binding site (PBS).  Reverse transcription is stimulated by other interactions between the tRNA molecule and sequences in the viral genome. A detailed interaction model was proposed based on biochemical probing studies, in which an A-rich loop in the HIV-1 genome interacts with the U- rich anticodon loop of the tRNA^lys3 primer.  Recently, a novel motif was identified in the HIV- 1 genome, which is critical for initiation and processive elongation of reverse transcription. This motif was termed the Primer Activation Signal (PAS) and interacts with a sequence in the TΨC arm of the tRNA^lys3 primer. Interestingly, the PAS enhancer element is masked in the viral transcript through base-pairing, and reverse transcription can be activated by mutational exposure of the PAS. The presence of the PAS enhancer element in a repressive RNA structure may provide a mechanism for positive and negative regulation of HIV-1 reverse transcription. Retroviral reverse transcription appears a highly specific process that is regulated by multiple interactions between the tRNA primer and the viral RNA genome.