We have analyzed the sequence-specific interaction between the Escherichia coli tryptophan (Trp) repressor and its operator using challenge phage vectors. These phages, derivatives of Salmonella phage P22 that have substitutions of synthetic, symmetric trp operators for the P22 rant operator, provide a genetic assay for DNA binding in vivo. Phages carrying operators that retain the determinants of Trp repressor binding efficiently lysogenize cells producing repressor; in contrast, phages with operators missing critical determinants kill such hosts. The binding determinants revealed by this assay corroborate a simple docking model for the Trp repressor-operator interaction postulated from the repressor crystal structure, and account for both the specificity of repressor binding and the ability of Trp repressor to recognize multiple, tandem DNA sites.