The structure of herpes simplex virus (HSV-1) replicative DNA was studied employing pulse-field gel electrophoresis to allow separation and direct visualization of large size virus DNA fragments. Accumulation of HSV-1 DNA that fails to enter the gel was detected from 6 hr postinfection. This corresponds, at least in part, to high molecular weight replicative intermediates. Analysis of this DNA employing restriction enzymes SpeI and AseI, which cut only once and twice respectively in the HSV-1 genome, revealed that these intermediates are concatemeric and contain adjacent genomes with L components in different orientations. Adjacent genomes with L components in the same orientation or with L components in inverted orientations were found in similar amounts, suggesting that inversions occur frequently. Our results suggest that inversions of L components are generated by a quite efficient mechanism, perhaps during replication. Observations made in this work could be explained by rolling circle-based models of replication of HSV-1 genomes, with replication starting at the different origins and/or combined with strand transfer at inverted repeats.