During viral replication, HIV-1 reverse transcriptase (RT) plays a pivotal role in converting genomic RNA into proviral DNA. While the biologically relevant form of RT is the p66-p51 heterodimer, two recombinant homodimer forms of RT, p66-p66 and p51-p51, are also catalytically active. Here we investigate the binding of the three RT isoforms to a fluorescently labeled 19/50-nucleotide primer/template DNA duplex by exploiting single-molecule protein-induced fluorescence enhancement (SM-PIFE). PIFE, which does not require labeling of the protein, allows us to directly visualize the binding/unbinding of RT to a double-stranded DNA substrate. We provide values for the association and dissociation rate constants of the RT homodimers p66-p66 and p51-p51 with a double-stranded DNA substrate and compare those to the values recorded for the RT heterodimer p66-p51. We also report values for the equilibrium dissociation constant for the three isoforms. Our data reveal great similarities in the intrinsic binding affinities of p66-p51 and p66-p66, with characteristic Kd values in the nanomolar range, much smaller (50-100-fold) than that of p51-p51. Our data also show discrepancies in the association/dissociation dynamics among the three dimeric RT isoforms. Our results further show that the apparent binding affinity of p51-p51 for its DNA substrate is to a great extent time-dependent when compared to that of p66-p66 and p66-p51, and is more likely determined by the dimer dissociation into its constituent monomers rather than the intrinsic binding affinity of dimeric RT.