Different passages of the vaccinia virus strain Ankara (CVA wild-type) during attenuation to MVA (modified vaccinia virus Ankara) have been analysed to detect alterations in the genome. Physical maps for the restriction enzymes HindIII and XhoI have been established. Six major deletions relative to the wild-type strain CVA could be localized. They reduce the size of the entire genome from 208 kb (CVA wild-type) to 177 kb for the MVA strain. Four deletions occurred during the first 382 passages and the resulting variant (CVA 382) displays an attenuated phenotype similar to that of the MVA strain. The deletions are located in both terminal fragments, affect two-thirds of the host range gene K1L and eliminate 3.5 kb of a highly conserved region in the HindIII A fragment. During the next 190 passages leading to MVA two additional deletions appeared. Again, one is located in the left terminal fragment, and the other includes the A-type inclusion body gene. Neither of the deletions appear to participate in further attenuation of the virus. Rescue of the partially deleted host range region with the corresponding wild-type DNA restored the ability of the attenuated strains MVA and CVA 382 to grow in some non-permissive tissue cultures. Nevertheless, the complete host range of the wild-type strain was not recovered. Also, plaque-forming behaviour and reduced virulence were not influenced. From the data presented it may be concluded that the partially deleted host range gene is not solely responsible for attenuation.