AbstractThe analysis of 23 clinical isolates of herpes simplex virus type 1 (HSV‐I) showed that 15 of 15 isolates that had undergone a few passages in tissue culture (fresh isolates) and two of eight isolates that had never been passaged (new isolates) were composed of a mixed population with respect to plaque morphology in Vero cells. Cloning and characterization of 10 large plaque viruses (L variants) and nine small plaque viruses (S variants), obtained from seven different isolates, showed the following. BamHI DNA restriction patterns of the L and the S variants from a single isolate differed only with respect to the electrophoretic mobility of the fragments that contain reiteration of specific sequences; they did not differ regarding the presence or the absence of restriction endonuclease cleavage sites. The L and the S variants differed with respect to the electrophoretic profiles of infected cell glycoproteins, thermosensitivity of growth and plaquing efficiency at 39°C, and, at least in the case of the two couples of variants that we tested, pathogenicity for the mouse. The hypothesis that the L variants might arise from the S variants during in vitro replication is discuss