Escherichia coli UvrD is a 3'-5' superfamily 1A helicase/translocase involved in a variety of DNA metabolic processes. UvrD can function either as a helicase or only as an single-stranded DNA (ssDNA) translocase. The switch between these activities is controlled in vitro by the UvrD oligomeric state; a monomer has ssDNA translocase activity, whereas at least a dimer is needed for helicase activity. Although a 3'-ssDNA partial duplex provides a high-affinity site for a UvrD monomer, here we show that a monomer also binds with specificity to DNA junctions possessing a 5'-ssDNA flanking region and can initiate translocation from this site. Thus, a 5'-ss-duplex DNA junction can serve as a high-affinity loading site for the monomeric UvrD translocase, whereas a 3'-ss-duplex DNA junction inhibits both translocase and helicase activity of the UvrD monomer. Furthermore, the 2B subdomain of UvrD is important for this junction specificity. This highlights a separation of helicase and translocase function for UvrD and suggests that a monomeric UvrD translocase can be loaded at a 5'-ssDNA junction when translocation activity alone is needed.