A defective genotype of herpes simplex virus strain Angelotti (HSV ANG) accumulates in the course of controlled serial high MOI virus passages representing 50–60% of the total number of mature virions in the seventh of these passages. Defective HSV ANG significantly differs from other defective HSV genotypes described so far: the DNA of the defective particles has the same buoyant density as nondefective DNA. In contrast to non-defective HSV ANG DNA, it is not attacked by the restriction endonucleases Eco R I, Hpa I and Hind III. Defective virions strongly suppress the formation of progeny virus. They do not interfere, however, with the synthesis of HSV-specified thymidine (TdR) kinase.