A physical map of the bacteriophage T5 genome was constructed by ordering the fragments produced by cleavage of T5 DNA with the restriction endonucleases SalI (4 fragments), SmaI (4 fragments), BamI (5 fragments), and HpaI (28 fragments). The following techniques were used to order the fragments. (i) Digestion of DNA from T5 heat-stable deletion mutants was used to identify fragments located in the deletable region. (ii) Fragments near the ends of the T5 DNA molecule were located by treating T5 DNA with X exonuclease before restriction endonuclease cleavage. (iii) Fragments spanning other restriction endonuclease cleavage sites were identified by combined digestion of T5 DNA with two restriction endonucleases. (iv) The general location of some fragments was determined by isolating individual restriction fragments from agarose gels and redigesting the isolated fragments with a second restriction enzyme. (v) Treatment of restriction digests with X exonuclease before digestion with a second restriction enzyme was used to identify fragments near, but not spanning, restriction cleavage sites. (vi) Exonuclease III treatment of T5 DNA before restriction endonuclease cleavage was used to locate fragments spanning or near the natural T5 single-chain interruptions. (vii) Analysis of the products of incomplete restriction endonuclease cleavage was used to identify adjacent fragments. MATERIALS AND METHODS Bacteriophage. Wild-type T5 and the heat-stable deletion mutants T5st(0) and T5st(102) (13, 15) were grown on Escherichia coli B23. EcoRI endonuclease. Purification and assay conditions for EcoRI have been described (11). Sally endonuclease. Streptomyces albus G (from G. Hayward) was grown at 30°C with shaking in a medium containing, in 1 liter: 0.33 g of yeast extract (Difco), 0.33 g of beef extract (Difco), 0.67 g of tryptone (Difco), trace FeSO4, and 3.33 g of glucose. After 2 days of growth, the mycelia were harvested by centrifugation and suspended in 0.01 M Tris, pH 7.9, containing 0.01 M 2-mercaptoethanol (approximately 10 ml per 15 g [wet weight] of mycelia). The cells were disrupted by sonication. Cell debris was removed from the extract by low-speed (8,000 rpm for 15 min in a Sorvall SS34 rotor) and high-speed centrifugation (33,000 rpm for 90 min in a Beckman SW50.1 rotor). The high-speed supernatant was made 1.0 M in NaCl and applied to a 200-ml column 249