The Escherichia coli UvrD protein is a 3′ to 5′ superfamily 1 DNA helicase that functions in nucleotide excision repair and methyl-directed mismatch repair of DNA, as well as DNA replication of certain plasmids. UvrD uses the energy of ATP binding and hydrolysis to unwind double-stranded DNA (dsDNA) and translocate along single-stranded DNA (ssDNA) with biased 3′ to 5′ directionality. Single turnover pre-steady state DNA unwinding kinetics experiments have shown that the UvrD dimer is the active form of the helicase in vitro, although a UvrD monomer can translocate along ssDNA with the same directionality as used in unwinding. Crystal structures show that UvrD can exist in two dramatically different conformations, “open” in the apo state and “closed” when forming a complex with a 3′- ssDNA-dsDNA junction. The rotational orientations of the 2B domain differ in these two states by about 100 degrees. To study the conformational changes of the 2B domain, double cysteine mutants with one pair on 1B and 2B domains and another pair on 2A and 2B domains were constructed and labeled with a mixture of donor-acceptor fluorophores such that the movement of 2B domain results in either an increase or a decrease in FRET, depending on the positions of the labeled fluorophores. Our ensemble studies show that the 2B domain is in the closed conformation at low salt and swivels open at high salt in the absence of DNA. The open and closed conformations are in dynamic equilibrium. The binding of UvrD to ssDNA induces the open conformation of the 2B domain. The swiveling of the 2B domain is also coordinated with ATP binding and hydrolysis.(This research was supported by the NIH (GM 045948).)