We are reporting the physical location of parental DNA sequences in 28 recombinants produced by crossing herpes simplex viruses (HSV) 1 and 2. The parental crosses were of two kinds. In the first, temperature-sensitive mutants of HSV-1 and HSV-2 were crossed to produce wild-type recombinants. In the second, temperature-sensitive mutants of HSV-1 rendered resistant to phosphonoacetic acid were crossed with wild-type HSV-2, and recombinants that multiplied at nonpermissive temperature and were resistant to the drug were selected. The DNAs of the recombinants were mapped with XbaI, EcoRI, HpaI, HsuI, BglII, and, in some instances, KpnI restriction endonucleases. The results were as follows. (i) We established the colinear arrangements of HSV-1 and HSV-2 DNAs. (ii) There was extensive interchange of genomic regions, ranging from the exchange or the entire L of S component of HSV DNA to substitutions of regions within the same component. In some recombinants, the reiterated sequences ab and ac bracketing the L and S components of HSV DNA were heterotypic. Most recombinants grew well and showed no obvious defects. (iii) The number of crossover events ranged from one to as many as six. Although crossover events occurred throughout the DNA, some clustering of crossover events was observed. (iv) Analysis of recombinants permitted localization of several markers used in this study and appears to be a useful technique for marker mapping. (v) As previously reported, HSV DNA consists of four populations, differing in relative orientation of the L and S components. All recombinants could be displayed in one arrangement of L and S such that the number of crossover events was minimized. The data are consistent with the hypothesis that only one arrangement of the parental DNA participates in the generation of recombinants.