Vaccinia viruses LC16m0 and LC16m8 are temperature-sensitive and low-neurovirulent variants derived from the Lister (Elstree) (LO) strain. Analyses of genome DNAs by digestion with restriction endonucleases and cross-hybridization of the digested fragments revealed that LC16m0 and LC16m8 possess a new XhoI site in addition to the 14 XhoI sites of LO. This new site is located at about 12 X 10(6) daltons from the right terminal end. There was no significant difference in the genome structures between the LC16 variants and LO except the new XhoI site and their terminal fragments which were not identified in LO owing to their heterogeneity. With HindIII digested fragments, there was no difference among the three viruses. This complete mapping raised the possibility that the putative gene responsible for temperature sensitivity and neurovirulence is located at the region of the XhoI site found in LC16m0 and LC16m8.