Intrinsic protein fluorescence of reverse transcriptases from HIV-1 and HIV-2 provides a sensitive signal for monitoring the interaction of the enzymes with primer/template duplex molecules. Kd values for 18/36-mer DNA/DNA duplexes were found to be in the range of a few nanomolar (about 3 times higher for the enzyme from HIV-2 than for that from HIV-1). The quenching of protein fluorescence induced on binding primer/template, together with an increase in extrinsic fluorescence on interaction with primer/template containing a fluorescent nucleotide at the 3′-end of the primer, was used to investigate the kinetics of interaction with reverse transcriptase from HIV-1. The results can be explained in terms of a two-step binding model, with a rapid diffusion-limited initial association (kass = ca. 5 × 108 M−1 s−1) followed by a slow isomerization step (k = ca. 0.5 s−1). These (forward) rate constants are increased in the presence of a non-nucleoside inhibitor (S-TIBO) of HIV-1 reverse transcriptase, while the reverse rate constant for the second step is decreased, leading to an increase in affinity between the enzyme and primer/template by a factor of at least 10 when S-TIBO is bound. The results are discussed in terms of present knowledge of the structure of reverse transcriptase.