We prepared vaccinia virus variants by introducing part of the HindIII D fragment of the DNA of the parental Lister (LO) strain (temperature-resistant and forming large plaques and pocks) into the attenuated LC16m8 strain (temperature-sensitive and forming small plaques and pocks) by the use of a homologous recombination technique in vivo. Special attention was paid to the HindIII D fragment, since this fragment has an extra XhoI site in LC16m8 which is absent from LO. After HindIII D of LO was introduced as a calcium phosphate precipitate into rabbit kidney (RK13) cells which had been infected with LC16m8, five virus variants (LOTC-1 to LOTC-5) forming much larger plaques than LC16m8 were cloned. In LOTC-2, LOTC-4 and LOTC-5, the introduction of at least part of HindIII D of LO into the corresponding HindIII D region of the LC16m8 genome was apparent as judged by the disappearance of the XhoI site, whereas variants LOTC-1 and LOTC-3 retained the site. The biological characteristics of all the LOTC variants were similar to each other. Their plaque size and pock size were similar to those of LO, whereas they were rather akin to LC16m8 with regard to temperature sensitivity and neurovirulence. The present results strongly suggested that part of the HindIII D fragment was involved in determining biological characteristics affecting plaque size and pock size, but had little influence on temperature sensitivity and neurovirulence.