We have investigated the role of sequences that surround the primer binding site (PBS) in the reverse transcriptase-mediated initiation of (-) strand DNA synthesis in human immunodeficiency virus type 1. In comparisons of reverse transcription initiated from either the cognate primer tRNALys.3 or a DNA primer D-Lys.3, bound to PBS sequences, we observed that a +3 pausing site occurred in both circumstances. However, the initiation reaction with tRNALys.3 was also characterized by a pausing event after incorporation of the first nucleotide. Alteration of sequences at the 5'-end instead of the 3'-end of the PBS resulted in elimination of the +3 pausing site, suggesting that this site was template sequence-dependent. In contrast, the pausing event at the +1 nucleotide position was still present in experiments that employed either of these mutated RNA templates. The mutations at the 5'-end of the PBS also caused a severely diminished rate of initiation and the strong arrest of reactions at the +1 stage when tRNALys.3 was used as primer. Therefore, we propose that the +1 pausing event is an initiation-specific event in regard to reactions primed by tRNALys.3 and that sequences at the 5'-end of the PBS may facilitate the release of reverse transcription from initiation to elongation.