Abstract A detailed restriction endonuclease map of Escherichia coli phage N4 DNA is presented. The cleavage sites for restriction endonucleases Hae II (1 cut), Hha I (3 cuts), Xba I (5 cuts), Ava II (15 cuts), and Hpa I (25 cuts) have been determined by (1) digestion with endonucleases after end labeling of the DNA; (2) digestion with two endonucleases; (3) digestion of N4 DNA after λ exonuclease treatment; and (4) annealing of isolated fragments to nitrocellulose blots containing fragments obtained by digestion with a different restriction endonuclease. N4 DNA was determined to contain 71 kilobase pairs. Cross-hybridization of the terminal fragments suggested the presence of terminal repetition. This observation was confirmed by electron microscopy of N4 DNA molecules annealed after λ exonuclease digestion. N4 DNA contains direct terminal repeats approximately 500 base pairs in length.