DNA from the WR strain of vaccinia virus was cleaved with five restriction endonucleases and the molecular weight of each restriction fragment was determined. From a summation of the molecular weights of these DNA fragments, the molecular weight of the vaccinia genome was estimated to be approximately 130 × 106. By electrophoresis in agarose gels under alkaline conditions, two HindIII fragments (WR-HindIII-34.8, WR-HindIII-22.7) and two SalI fragments (WR-SaI-3.5 and either a WR-SalI-1.1 fragment or WR-Sall-0.9) were identified as arising from the cross-linked terminal regions of the vaccinia virus genome. Heterogeneity in the size of these two fragments was observed upon cleavage of DNA purified from vaccinia virus (WR strain) which was serially passaged in various cell lines, but not in plaque-purified virus preparations. The structure of DNA from the CV-1 strain of vaccinia virus was also analyzed by cleavage with restriction endonucleases and compared to that of the WR strain; with the exception of the terminal fragments, all HindIII fragments observed in the WR digest were also observed in the CV-1 digest. Among the digestion products of vaccinia DNA, we have observed restriction fragments which are present in submolar quantities. The presence and size of these bands appear to depend upon the host cell in which the virus is propagated.