Non-glucosylated (Glu-) T-even phage DNAs are restricted by Escherichia coli RgIA and RgIB endonucleases with different specificities. RgIB endonuclease activity is strongly inhibited by anti-restriction endonuclease (Arn) encoded by the bacteriophage T4 genome. The nucleotide sequence of the arn gene encoding Arn was determined. The product of the cloned arn gene was overexpressed by the T7 RNA polymerase/promoter system, and its molecular size is consistent with that predicted from the open reading frame of the arn gene. The arn gene is located between the asiA gene and motA gene in the region of 161,300-161,578 nucleotides.