This paper concerns the properties of herpes simplex virus 1 DNA replicating in HEp-2 and human embryonic lung cells. The results were as follows. (i) Only a small fraction of input viral DNA entered the replicative pool. The bulk of the input viral DNA cosedimented with marker viral DNA and did not appear to be degraded or dissociated into L and S components. (ii) Nascent DNA sedimented faster and banded at a higher density than that of mature viral DNA extracted from virions. Pulse-chase experiments indicated that nascent DNA acquires the sedimentation rate and buoyant density of viral DNA within 30 to 40 min after its synthesis. (iii) Electron microscopic studies indicated that the DNA extracted from cells replicating viral DNA and banding at the density of viral DNA contained: (a) linear, full-size molecules with internal gaps and single-stranded regions at termini; (b) molecules with lariats, consisting of a linear segment up to 2x the size of mature DNA and a ring ranging from 0.5 x 10(6) to 100 x 10(6) in molecular weight, showing continuous and discontinuous forks; (c) circular, double-stranded molecules, both full-size and multiples of 18 x 10(6) in molecular weight, but without forks or loops; (d) molecules showing "eye" and "D" loops at or near one end of the DNA; (e) large, tangled masses of DNA, similar to those observed for T4 and pseudorabies virus replicating DNAs, containing loops and continuous and discontinuous forks. The electron micrographs are consistent with the hypothesis that the single-stranded ends on the DNA anneal to form a hairpin, that the DNA synthesis is initiated at or near that end and proceeds bidirectionally to form a lariat, and that resulting progeny derived by semiconservative replication are "head-to-head" and "tail-to-tail" dimers.