The kinetic mechanism by which Escherichia coli RecBCD helicase unwinds duplex DNA was studied using a fluorescence stopped-flow method. Single turnover DNA unwinding experiments were performed using a series of fluorescently labeled DNA substrates containing duplex DNA regions ranging from 24 bp to 60 bp. All or no DNA unwinding time courses were obtained by monitoring the changes in fluorescence resonance energy transfer between a Cy3 donor and Cy5 acceptor fluorescent pair placed on opposite sides of a nick in the duplex DNA. From these experiments one can determine the average rates of DNA unwinding as well as a kinetic step-size, defined as the average number of base-pairs unwound between two successive rate-limiting steps repeated during DNA unwinding. In order to probe how the kinetic step-size might relate to a mechanical step-size, we performed single turnover experiments as a function of [ATP] and temperature. The apparent unwinding rate constant, kUapp, decreases with decreasing [ATP], exhibiting a hyperbolic dependence on [ATP] (K 1/2=176(±30)μM) and a maximum rate of kUapp= 204(±4)stepss-1 (mkUapp=709(±14)bps -1) (10 mM MgCl2, 30 mM NaCl (pH 7.0), 5% (v/v) glycerol, 25.0°C). kUapp also increases with increasing temperature (10-25°C), with Ea=19(±1)kcalmol-1. However, the average kinetic step-size, m=3.9(±0.5)bpstep-1, remains independent of [ATP] and temperature. This indicates that even though the values of the rate constants change, the same elementary kinetic step in the unwinding cycle remains rate-limiting over this range of conditions and this kinetic step remains coupled to ATP binding. The implications of the constancy of the measured kinetic step-size for the mechanism of RecBCD-catalyzed DNA unwinding are discussed. © 2004 Elsevier Ltd. All rights reserved.