The Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA in a reaction that is coupled to ATP binding and hydrolysis. The Rep protein is a stable monomer in the absence of DNA but dimerizes upon binding either single-stranded or duplex DNA, and the dimer appears to be the functionally active form of the Rep helicase. As a first step toward understanding how ATP binding and hydrolysis are coupled energetically to DNA unwinding, we have investigated the kinetic mechanism of nucleotide binding to the Rep monomer (P) using stopped-flow techniques and the fluorescent ATP analogue, 2′(3′)-O-(N-methylanthraniloyl-ATP (mantATP). The fluorescence of mantATP is enhanced upon Rep binding due to energy transfer from tryptophan. The results are consistent with the following two-step mechanism, in which the bimolecular association step is followed by a conformational change in the P-mantATP complex: (formula ommited) The following rate and equilibrium constants were determined at 4 °C in 20 mM Tris·HCl (pH 7.5), 6 mM NaCl, 5 mM MgCl2, and 10% (v/v) glycerol: k+1 = (1.1 ± 0.2) × 107 M−1 s−1; k−1 = 3.2 (±0.5) s−1; k+2 = 2.9 (±0.5) s−1; k−2 = 0.04 (±0.005) s−1; K1 = k+1/k−1 = (3.4 ± 0.8) × 106 M−1; K2 = k+2/k−2 = 73 (±10); Koverall = K1K2 = (2.30 ± 0.6) × 108 M−1. Similar rate and equilibrium constants are obtained with mantATPγS, whereas the apparent rate constant for mantAMPPNP binding is 15-fold lower than for mantATP and equilibrium binding is weaker (Koverall ∼ 106 M−1). Rep monomer does bind mantATP in the absence of Mg2+ (Koverall∼ 5 × 105 M−1), although the four rate constants in the above reaction increase by at least 8-fold (k−1 and k−2 increase by ∼100- and ∼ 1000-fold, respectively). The affinities of Mg2+ for P‒mantATP and (P‒mantATP)* are 10- and 1000-fold higher than those for nucleotide-free Rep monomer, indicating that the second step in the reaction is associated with a marked increase in Mg2+ affinity. The bound Mg2+ in a (P‒mantATP)*‒Mg2+ complex dissociates at a rate that is comparable to the rate of mantATP release. Single-turnover kinetic studies with the Rep monomer indicate a low, but significant, DNA-independent ATPase activity, with a first-order cleavage rate constant of ∼10−3 s−1 at 4 °C, which increases with temperature (Eact = 18 ± 2 kcal mol−1). These results indicate that Rep is a DNA-stimulated ATPase rather than a DNA-dependent ATPase. The absence of a burst of ADP formation in a multiple ATP turnover experiment suggests that product release is not rate-limiting under these conditions. The approaches described here and in the accompanying paper (Moore & Lohman, 1994) will be useful for subsequent studies of the more complex Rep dimeric species and of other helicases.