Serial undiluted passage of clonally purified herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in 4 different types of cells resulted in (i) partial loss (84–98%) of infectivity and (ii) the appearance of a new DNA within the infected cells with a higher buoyant density in CsCl (p = 1.732 g/cm3) than that of HSV-1 (1.725 g per cm3) or that of HSV-2DNA (1.727 g/cm3). Serial diluted passages of these stocks restored the high infectivity titers with a concomitant loss of the high density DNA within the infected cells. Serologic studies and electron microscopy indicated that the HSV stocks were not contaminated with an extraneous virus which might yield a DNA with the high buoyant density. Characterization of purified HSV from the serially passed undiluted stocks indicates that the high density DNA is a part of the HSV virion replicating under these conditions. The results obtained suggest the production of defective interfering particles after undiluted passage of HSV.