The formation of defective herpes simplex virus (HSV) in BSC-1 cells and the synthesis of defective virus DNA was studied. The fourth consecutive passage of undiluted virus yielded defective DNA that was 0.008 g/ml more dense than wild type (w.t.) virus DNA. The amount of defective DNA increased at passage 6 concomitantly with the decrease in infectious virus progeny. The synthesis of defective DNA was always accompanied by w.t. virus DNA synthesis. Defective DNA from both infected nuclei and defective virions had a mol. wt. of 100 X 10(6) and was linear as determined by electron microscopy. Electron microscopy of defective virus DNA at passage 6 revealed circular molecules varying in size in addition to linear DNA molecules with the length of intact virion DNA. The circular DNA molecules had contour lengths of 10, 5, 2.5 and less than 2.5 micron. The smallest circular DNA molecules had a contour length of 0.3 micron, possibly one virus gene. In addition, circular-linear DNA molecules were observed in which both the circular and the linear components varied in length. Most of these DNA molecules had circular components of either 2.5 or 5.0 micron, and linear components varying in length from less than 1 to 50 micron. Based on the present study, it is proposed that the S component of w.t. virus DNA is fragmented into small circular molecules that serve as templates for DNA synthesis, possibly by the rolling circle mechanism.