The initiation of (-) strand DNA synthesis by HIV-1 reverse transcriptase was examined using a transient kinetic approach and a physiologically relevant RNA 18-mer/RNA 36-mer primer-template substrate. HIV-1 reverse transcriptase (RT) was found to bind with reasonably high affinity to the RNA/RNA substrate (K(d) = 90 nM), although the affinity for DNA/RNA and DNA/DNA substrates is higher (K(d) approximately 5 nM). A pre-steady-state burst of deoxynucleotide incorporation (k(obsd) = 1.0 s(-)(1)) into the RNA duplex was observed followed by a slower steady-state release of the elongated primer-template product (k(ss) = 0.58 s(-)(1)). The observation of a burst provides evidence that the release of the product is most likely the rate-limiting step in the overall kinetic pathway for the enzymatic reaction during a single deoxynucleotide incorporation event. Furthermore, the release of this product was 5-fold faster than that for elongated DNA/RNA and DNA/DNA products. Single-turnover experiments showed that there is a hyperbolic dependence of the rate of deoxynucleotide incorporation on the concentration of dCTP and demonstrated that the maximum rate of dCTP incorporation (k(pol) = 1.4 s(-)(1)) is 33- and 12-fold slower than the values for DNA/RNA and DNA/DNA primer-template substrates, respectively, while the affinity of dCTP (K(d) = 780 microM) for the HIV-1 RT.RNA/RNA complex is 56- and 71-fold weaker than the affinities for HIV-1 RT.DNA/RNA and HIV-1 RT.DNA/DNA complexes, respectively. Consequently, the overall efficiency of dCTP incorporation (k(pol)/K(d)) into the RNA/RNA substrate is approximately 1800- and 800-fold less than that for DNA/RNA and DNA/DNA substrates, respectively. These findings provide evidence which suggests that the HIV-1 RT.RNA/RNA.dCTP ternary complex exists in a significantly different conformation compared to ternary complexes involving DNA/RNA and DNA/DNA substrates. A model summarizing these results is presented, and implications for the molecular mechanism of initiation of (-) strand DNA synthesis by RT are discussed.