By means of a nitrocellulose filter binding assay, DNA binding activities among proteins fractionated from extracts of Escherichia coli carrying lambda dv have been surveyed. An activity was found that binds specifically to a fragment of 164 base pairs that specifies the lambda replication origin (lambda ori). This activity was not detected in an extract of cells not carrying the lambda dv plasmid. The activity was detected in extracts of cells carrying a hybrid plasmid in which the entire lambda O gene had been cloned and placed under the control of the lac promoter. Deletion of a 60 base pair segment in the 'amino-terminal region' of the O gene abolished this activity, indicating that the lambda ori binding protein is coded for by the lambda O gene. The ori-specific binding protein was purified by five fractionation steps. The most purified preparation consists of a major polypepide that migrates with a molecular weight of 32,000 in SDS-polyacrylamide gel electrophoresis. Binding of O protein to ori occurs in the absence of other protein aceous components.