Three non-engineered, spontaneously occurring herpes simplex virus type 1 (HSV-1) mutants (GN52, GN82, and GN91) that have a deletion of approximately 10 kbp (including a part of the UL55 gene, the entire UL56 gene, and one copy of the inverted repeat sequences of the L component (R(L))) and retain the a sequence were isolated. The yields of the mutants at 24 h post-adsorption in cultured cells were comparable to that of an HSV-1 isolate (GN28) without the deletion. Although the three mutants lost one copy of R(L), the L component in replicative intermediates of the mutants inverted. DNA replicative intermediates of the three mutants were flanked by the L component, like those of GN28. The three mutants were generated through recombination involving regions around the authentic cleavage site in the a sequence, suggesting an important role of the a sequence in the diversification of herpesviruses.