HIV-1 reverse transcriptase (RT) utilizes RNA oligomers to prime DNA synthesis. The initiation of reverse transcription requires specific interactions between HIV-1 RNA, primer tRNA3/(Lys), and RT. We have previously shown that extension of an oligodeoxyribo-nucleotide, a situation that mimicks elongation, is unspecific and differs from initiation by the polymerization rate and dissociation rate of RT from the primer-template complex. Here, we used replication intermediates to analyze the transition from the initiation to the elongation phases. We found that the 2'-hydroxyl group at the 3' end of tRNA had limited effects on the polymerization and dissociation rate constants. Instead, the polymerization rate increased 3400- fold between addition of the sixth and seventh nucleotide to tRNA3/(Lys). The same increase in the polymerization rate was observed when an oligoribonucleotide, but not an oligodeoxyribonucleotide, was used as a primer. In parallel, the dissociation rate of RT from the primer-template complex decreased 30-fold between addition of the 17th and 19th nucleotide to tRNA3/(Lys). The polymerization and dissociation rates are most likely governed by interactions of the primer strand with helix αH in the p66 thumb subdomain and the RNase H domain of RT, respectively.