Attenuated variants of vaccinia virus with two specific and stable genetic markers were obtained after long-term passage of persistently infected Friend erythroleukemia (FEL) cells. The genetic markers were an 8-MDa deletion on the left HindIII-C terminus of the viral genome and sequence alterations localized in the middle of the HindIII-A DNA fragment. This latter genetic marker led to small plaque size phenotype of these variants. The mode of replication of these variants in tissue culture cells and their virulence in mice were analyzed. In cultured cells, these variants have greatly reduced virus yields in cell lines of different origins. These variants penetrate into cells, synthesize early and late viral proteins, and replicate their DNA with about the same efficiency as wild-type virus. The defect of these variants appears at some step during virus maturation. When groups of BALB/c mice were inoculated intraperitoneally (ip) with these variants, 50% of the mice survived with greater than or equal to 1 X 10(9) plaque-forming units (PFU) as opposed to about 50% survival for mice inoculated with 1 X 10(6) PFU of wild-type virus. Mice inoculated with these variants were fully protected when challenged ip with lethal doses of wild-type virus. The reduced virulence of these variants correlated with the 8-MDa deletion; in addition, the plaque size phenotype marker contributes to a further decrease of the virulence of vaccinia virus. Due to their limited virus production and protective immune response, these variants may be potentially useful as vaccines.