Restriction site mapping of the genomes of eight different isolates of adenovirus serotype 7 (Ad7) has been performed with six different restriction endonucleases. In this analysis, 37 different restriction sites were localized. Three distinctly different cleavage patterns of the genomes of the Ad7 strains were observed. These strains could not be distinguished by serological techniques. The following three subtypes were defined on the basis of their restriction site patterns: the Ad7 prototype, represented by strain Gomen originally isolated from a case of pharyngitis; subtype Ad7a, represented by the Ad7 vaccine strain and strains isolated from undifferentiated respiratory disease and from a healthy carrier; and a third subtype of Ad7, represented by three strains which were isolated from Swedish patients, all having pronounced clinical symptoms indicating severe systemic infection. A comparison of the restriction site maps of the protype of Ad3 and the three subtypes of Ad7 indicated greater differences in the position of restriction sites between strains of Ad7 than between strains of the two serotypes. This technique is consequently recommended to obtain a more precise definition of distinct entities of viruses.