Reverse transcription is an essential step in the retroviral life cycle, as it converts the genomic RNA into DNA. In this review, we describe recent developments concerning the initiation step of this complex, multi-step reaction. During initiation of reverse transcription, a cellular tRNA primer is placed onto a complementary sequence in the viral genome, called the primer binding site or PBS. The viral enzyme reverse transcriptase (RT) recognizes this RNA-RNA complex, and catalyzes the extension of the 3′ end of the tRNA primer, with the viral RNA (vRNA) acting as template. The initiation step is highly specific and most retroviruses are restricted to the use of the cognate, self-tRNA primer. Human immunodeficiency virus type 1 (HIV-1) uses the cellular tRNALys,3 molecule as primer for reverse transcription. No spontaneous switches in tRNA usage by HIV-1 or other retroviruses have been described and attempts to change the identity of the tRNA primer were unsuccessful in the past. These observations indicate that the virus strongly prefers the self-primer, suggesting that a very specific mechanism for primer selection must exist. Indeed, tRNA primers are selectively packaged into virus particles, are specifically recognized by RT and are placed onto the viral RNA genome via base pairing to the PBS and other sequence motifs, thus rendering a specific initiation complex. Analysis of this critical step in the viral life cycle may result in the discovery of novel antiviral drugs in the battle against HIV/AIDS.