We have analyzed the efficiency and accuracy of polymerization at several different stages during the initiation of human immunodeficiency virus type 1 (HIV-1) (+)-strand DNA synthesis. This reaction is of particular interest, as it involves the recruitment by reverse transcriptase of an RNA primer that serves as substrate for both the polymerase and RNase H activities of the enzyme. We found that the correct incorporation of the first two nucleotides was severely compromised and that formation of mismatches was completely absent at this stage of initiation. Although the fidelity of incorporations decreased concomitantly with ensuing polymerization, the elongation of mispaired primers was literally blocked. Instead, mispaired primer strands initiated a switch from active synthesis of DNA to premature RNase H-mediated primer removal. These findings suggest the existence of a fragile equilibrium between these two enzymatic activities that is shifted toward RNase H cleavage once the polymerization process is aggravated. Our data show that the initiation of HIV-1 (+)-strand DNA synthesis differs significantly from reactions involving other primer/template combinations, including tRNA-primed (-)-strand DNA synthesis.