The bacteriophage P22-based challenge phage system was used to study lambda integrase (Int) protein binding to its arm-type recognition sequences in the bacteriophage lambda attachment site. Challenge phages were constructed that carried inserts containing either the contiguous P'123 arm-type sites or the single P'1 site within the P22 phage promoter, Pant, which is required for expression of antirepressor. If Int protein binds to these sequences in vivo, it represses transcription from Pant. We found that Int repressed Pant in phages carrying the P'123 sites more efficiently than those carrying only the P'1 site, suggesting that the protein binds cooperatively at the three adjacent sites. The Int protein from a related lambdoid phage, HK022, also repressed transcription by binding to the same arm-type sites. Mutations in the P'123 or P'1 sites that impair Int binding were isolated by selecting mutant phages that express antirepressor in the presence of Int. DNA sequence analyses showed that most of the mutants in the challenge phages carrying the P'123 sites contained multiple changes and that two mutants contained only single-base-pair changes at positions that are completely conserved among all arm-type sites. Thirty-five mutants were isolated and analyzed from phages containing only the P'1 site. Most mutants contained single-nucleotide changes, and mutations were isolated at 8 of the 10 positions of the site, suggesting that most if not all base pairs in the conserved recognition sequence are involved in Int binding.