The kinetic mechanism by which the DNA repair helicase UvrD of Escherichia coli unwinds duplex DNA was examined with the use of a series of oligodeoxynucleotides with duplex regions ranging from 10 to 40 base pairs. Single-turnover unwinding experiments showed distinct lag phases that increased with duplex length because partially unwound DNA intermediate states are highly populated during unwinding. Analysis of these kinetics indicates that UvrD unwinds duplex DNA in discrete steps, with an average "step size" of 4 to 5 base pairs (approximately one-half turn of the DNA helix). This suggests an unwinding mechanism in which alternating subunits of the dimeric helicase interact directly with duplex DNA.