Escherichia coli DNA polymerase IV, encoded by the dinB gene, is a member of the Y family of specialized DNA polymerases. Pol IV is capable of synthesizing past DNA lesions and may help to restart stalled replication forks. However, Pol IV is error-prone, contributing to both DNA damage-induced and stress-induced (adaptive) mutations. Here we demonstrate that Pol IV interacts in vitro with Rep DNA helicase and that this interaction enhances Rep's helicase activity. In addition, Pol IV polymerase activity is stimulated by interacting with Rep, and Pol IV β clamp-binding motif appears to be required for this stimulation. However, neither Rep's helicase activity nor its ability to bind DNA is required for it to stimulate Pol IV's polymerase activity. The interaction between Rep and Pol IV is biologically significant in vivo as Rep enhances Pol IV's mutagenic activity in stationary-phase cells. These data indicate a new role for Rep in contributing to Pol IV-dependent adaptive mutation. This functional interaction also provides new insight into how the cell might control or target Pol IV's mutagenic activity.