Previous pre-steady-state kinetic studies of equine infectious anemia virus-1 (EIAV) reverse transcriptase (RT) showed two effects of DNA substrates containing the central termination sequence (CTS) on the polymerization reaction: reduction of burst amplitude in single nucleotide addition experiments and accumulation of termination products during processive DNA synthesis [Berdis, A. J., Stetor, S. R., Le Grice, S. F. J., and Barkley, M. D. (2001) Biochemistry 40, 12140-12149]. The present study of HIV RT uses pre-steady-state kinetic techniques to evaluate the molecular mechanisms of the lower burst amplitudes using both random sequence and CTS-containing DNA substrates. The effects of various factors, including primer/template length, binding orientation, and protein concentration, on the burst amplitude were determined using random sequence DNA substrates. The percent active RT increases with total RT concentration, indicating that reversible dissociation of RT dimer is responsible for substoichiometric burst amplitudes with normal substrates. This finding was confirmed by gel mobility shift assays. Like EIAV RT, HIV RT showed lower burst amplitudes on CTS-containing DNA substrates compared to random sequences. The dissociation kinetics of RT-DNA complexes were monitored by enzyme activity and fluorescence. Biphasic kinetics were observed for both random sequence and CTS-containing DNA complexes, revealing two forms of the RT-DNA complex. A mechanism is proposed to account for reduction in burst amplitude of CTS-containing DNA that is consistent with the results of both single nucleotide addition and dissociation experiments. The two forms of the RT-DNA complex may represent partitioning of primer/template between the P- and N-sites on RT for the nucleic acid substrate.