We have identified two functions for RecA-mediated ATP hydrolysis during DNA strand exchange. First, ATP hydrolysis renders RecA protein-mediated DNA strand exchange unidirectional (5' to 3' with respect to the single-stranded DNA). In the presence of a nonhydrolyzable analog adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), DNA strand exchange is bidirectional. Second, ATP hydrolysis is required for extensive formation of hybrid DNA. In the presence of ATP hydrolysis, the length of the exchanged region is limited only by the available homology, whereas in the absence of ATP hydrolysis, only 2 kilobase pairs or less of hybrid DNA are formed before branch migration is blocked in the majority of paired intermediates. Both of these functions of RecA protein-mediated ATP hydrolysis are crucial in ensuring the effectiveness of recombinational DNA repair, especially when the lesion to be repaired is distant from the initial crossover point.