A central feature of retrovirus replication is integration of the provirus into host cell DNA, but the specificity of this step for cell target sequences has not been clarified. To investigate this issue, we developed a method for screening and comparing large numbers of unselected integration events. Using a replication-competent Rous sarcoma virus containing a bacterial suppressor tRNA gene as a selectable marker, we obtained collections of clones comprising integrated provirus together with host flanking sequences. Hybridization and sequence analysis of the flanking sequence reveals the presence of a number of strongly preferred integration targets. Within these targets, independent integration events occur at sites identical to the base.