The mechanism by which Escherichia coli RecBCD DNA helicase unwinds duplex DNA was examined in vitro using pre-steady-state chemical quenched-flow kinetic methods. Single turnover DNA unwinding experiments were performed by addition of ATP to RecBCD that was prebound to a series of DNA substrates containing duplex DNA regions ranging from 24 bp to 60 bp. In each case, the time-course for formation of completely unwound DNA displayed a distinct lag phase that increased with duplex length, reflecting the transient formation of partially unwound DNA intermediates during unwinding catalyzed by RecBCD. Quantitative analysis of five independent sets of DNA unwinding time courses indicates that RecBCD unwinds duplex DNA in discrete steps, with an average unwinding "step-size", m = 3.9(±1.3) bp step-1, with an average unwinding rate of kU = 196(±77) steps s-1 (mkU = 790(±23) bp s-1) at 25.0°C (10 mM MgCl2, 30 mM NaCl (pH 7.0),) 5% (v/v) glycerol). However, additional steps, not linked directly to DNA unwinding are also detected. This kinetic DNA unwinding step-size is similar to that determined for the E. coli UvrD helicase, suggesting that these two SF1 superfamily helicases may share similar mechanisms of DNA unwinding. © 2002 Elsevier Science Ltd. All rights reserved.