Many facets of herpes simplex virus (HSV) DNA replication are not understood and advances in our knowledge depend on accurate characterization of high-molecular-weight replicative intermediates. In the present work, we have used a refinement of field-inversion gel electrophoresis (FIGE) to analyze infected-cell DNA. Infected Vero cells were encapsulated and manipulated in agarose microbeads, allowing intact replicative intermediates to be recovered easily from the wells of FIGE gels after electrophoretic removal of 152-kb linear viral genomes. Digestion of replicative intermediates with SpeI, which cuts the viral genome once, generated two novel DNA fragments (186 and 118 kb), in addition to the expected unit-length fragment (152 kb) predicted to arise from head to tail concatemers generated by rolling-circle replication. The SpeI fragments are the products of previously unidentified concatemers containing a head to tail arrangement of different HSV isomers, with respect to the orientation of the long segment of the viral genome. Such concatemers were prominent at an early stage of DNA synthesis when replicating DNA appeared still to be in a circular configuration, raising the possibility that isomerization of the viral genome is intimately linked to the initial round of DNA replication. Moreover, high-molecular-weight replicative intermediates were flanked exclusively by the long segment of the viral genome, indicating a unique initiation/termination or cleavage/packaging mechanism during HSV DNA replication and viral maturation.