We studied the effects of human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein on the kinetics of annealing of nucleic acids using model substrates derived from the 3' end of the HIV-1 minus-strand strong-stop DNA (-sssDNA). We used HIV-1 reverse transcriptase (RT) to monitor the annealing reaction. Using several different DNA primers and acceptor oligonucleotides, we found that the rate of annealing increased with the size of the complementary region of the primer and the acceptor strands and decreased when secondary structures could be formed in either the primer or the acceptor strands. The secondary structure had a larger effect on the rate of annealing if the secondary structure extends to the 3' end of the nucleic acid(s). NC protein reduced the rate of annealing between strands with short homologies. NC had no major effect on the rate of annealing when there were at least 13 bases of complementarity between the primer and the acceptor strands and neither strand could form a stable secondary structure. NC increased the rate of annealing when the primer and/or the acceptor strand could form a secondary structure in the region of complementarity. When two strands were in competition as acceptors in an annealing reaction, the specificity of the annealing was determined by the length of the complementarity between the primer and the acceptor strands, the presence or the absence of secondary structures in the primer and/or the acceptor strand, and the presence or the absence of NC in the reaction. This suggests that NC facilitates strand transfer where the nucleic acids have considerable secondary structure (for example, the first strand transfers for viruses whose genomes have considerable secondary structure at their 3' ends). However, NC also appears to increase the fidelity of recombination by reducing strand transfers between segments that have limited complementarity.