An attempt was made to classify herpes simplex virus type 1 (HSV-1) isolates into subtypes on the basis of the combination of the gain or loss of specific cleavage sites of HSV-1 genomes with each of three restriction endonucleases (Bam HI, Kpn I, and Sal I). According to the criteria we used for the determination of HSV-1 subtypes, 93 strains of HSV-1 that were isolated in three areas of Japan (Sapporo, Tottori, and Kagawa) were tentatively classified into eight subtypes: subtypes A-H. The bulk of the strains (84 of 93) fell into three subtypes: A, C, and H. There were highly significant differences (P less than .01) in the proportion of subtypes A and H that were isolated in Sapporo as compared with those isolated in Tottori and in Kagawa, which are geographically far from Sapporo. No significant differences, however, were found in subtypes isolated in Tottori as compared with those isolated in Kagawa, which are geographically close to each other. These data suggest that there might be a correlation between the genome structure of HSV-1 and the areas of their isolation in Japan.