The interactions of nucleotides with RepA hexameric helicase from plasmid RSF1010 have been examined using nucleotide analogues, TNP-ADP, TNP-ATP, and MANT-ADP. The binding of the analogues is accompanied by strong quenching of the protein fluorescence. A quantitative fluorescence titration method has been applied to analyze the interactions, independent of any assumptions of proportionality between the fluorescence quenching and the average degree of binding. The fluorescence quenching as a function of the average degree of binding is expressed by an empirical function that enables analysis of the data, without the necessity of determining quenching parameters for different complexes. At saturation, the RepA hexamer binds six nucleotide molecules, indicating that each subunit of the hexamer can engage in interactions with the cofactor. The nucleotide macroscopic affinity decreases with the increasing degree of binding, indicating heterogeneity among the binding sites. A statistical thermodynamic hexagon model provides an excellent description of the binding process and requires only two interaction parameters, the intrinsic binding constant, K, and cooperativity parameter, σ. The heterogeneity in affinity reflects negative cooperative interactions between the binding sites. Analyses of the data provide clear evidence that the alternative model of two independent classes of binding sites does not describe the nucleotide binding. Such a model cannot account for both, the binding isotherms and the dependence of the fluorescence quenching upon the degree of binding. Thus, cooperative interactions between the nucleotide-binding sites are an intrinsic property of the RepA helicase. The presence of the cooperative interactions indicates significant communication among the subunits of the helicase. © 2005 American Chemical Society.