The effect of RNA secondary structure on the kinetics of DNA synthesis catalyzed by HIV-1 RT was determined using a 66 nucleotide RNA template containing a stable 12 base pair hairpin structure. Prior to reaching the hairpin structure, the primer elongation by RT was fast and the kinetics of polymerization was not affected by the presence of the secondary structure. Once within the regions of template secondary structure, polymerization was much slower and RT paused at five distinct sites [Suo, Z., & Johnson, K. A. (1997) Biochemistry (manuscript submitted for publication)]. Kinetic analysis of single nucleotide incorporation at the pause sites showed polymerization occurred by both a fast phase (54-76 s-1) and a slow phase (0.07-0.4 s-1) during a single binding event. The biphasic kinetics suggests that the DNA substrates are initially bound in both productive and nonproductive states at the polymerase site of RT. The nonproductively bound DNA is slowly converted into a productive state without dissociation from the enzyme. At the pause sites, the enzyme amplitudes of the fast phase are small (4.0-15%) while the amplitudes of the slow phase are large (11-40%). In contrast, only the reaction at the fast phase was observed at the nonpause sites and the enzyme amplitudes were large (63-66%) although the nucleotide incorporation rates (62-78 s-1) are similar to the fast phase rates at the pause sites. These indicate that DNA substrates were bound predominantly nonproductively at pause sites and productively at nonpause sites. However, the overall binding affinity of DNA substrates was measured by the nitrocellulose-DEAE double filter binding assay, binding affinity at both pause sites and nonpause sites was similar (9-38 nM). This indicates that substrates are bound tightly at the large binding cleft of HIV-1, although they may not be productively bound at the polymerase active site. These results and those reported elsewhere [Suo, Z., & Johnson, K. A. (1997) Biochemistry (manuscript submitted for publication)] are consistent with a model in which, at pause sites, HIV-1 RT remains bound to DNA substrates waiting for the melting of the next stem base pair of template secondary structure. Upon melting of the stem base pair, polymerization to fill the open template site is fast and largely irreversible, allowing RT to read through the stable hairpin structures.