Intrinsic protein fluorescence has been used to study dimerization of the HIV-1 reverse transcriptase (RT). We observed a 25% increase of the tryptophan fluorescence of the enzyme during dissociation of the subunits induced by the addition of acetonitrile. Upon reassociation of the separated subunits, the original fluorescence emission of the heterodimer is restored. A two-state transition model for the RT dimerization process in which the dimers are in equilibrium with folded monomers is proposed. The free energy of dissociation was determined to be 12.2 (± 0.2) kcal mol. In the absence of Mg2+ ions a decrease of this value was observed, whereas the addition of a synthetic primer/template (18/36mer) results in an increase of dimer stability. Analyzing the effect of Mg2+ on the establishment of the binding equilibrium, a dramatic effect with a 100-fold acceleration of the association by the divalent ion was observed.