The isolation and some properties of a virulent bacteriophage of Salmonella typhimurium, MB78, which is morphologically, serologically, and physiologically unrelated to P22, are reported. The phage has a noncontractile long tail with partite ends. It cannot multiply in minimal medium in the presence of citrate. MB78-infected cells are, however, killed in such medium. This phage cannot grow in rifampin-resistant mutants of the host. The latent period of growth of this phage is much shorter than that of P22. Both sieA and sieB genes of the resident P22 prophage are required to exclude the superinfecting MB78 phage, whereas all temperate phages related to P22 are excluded by either one or both of the genes individually. Restriction endonuclease cleavage patterns of P22 and MB78 are distinctly different. The absence of homology between the two phages P22 and MB78 suggests that MB78 is not related to phage P22. In the course of studies with the temperate phages of Salmonella typhimurium, a virulent bacteriophage of the microorganism has been isolated. This phage is morphologically, serologically, and physiologically different from the temperate phage as well as the other virulent phage Felix 01 (13) of S. typhimurium reported so far. The present report deals with the purification and characterization of this new phage, which has been named MB78. MATERIALS AND METHODS [3H]thymidine (6,700 Ci/mol) was purchased from New England Nuclear Corp., Chicago, Ill. 32p in the form of orthophosphoric acid was the product of Bhabha Atomic Research Centre, Bombay, India. 2,5-Diphenyloxazole (PPO) and dimethyl 1,4-bis-(5-phenyloxazolyl)benzene (POPOP) were products of the Amersham/Searle Corp., Arlington Heights, Ill. Cesium chloride was obtained from Schwarz/Mann, Orangeburg, N.Y. All other chemicals were commercial preparations of analytical grade. Bacterial strains. S. typhimurium (LT2, strain 18)