Modified vaccinia virus Ankara (MVA), attenuated by over 500 passages in primary chick embryo fibroblasts (CEF), is presently being used as a safe expression vector. We compared the host ranges of MVA and the parental Ankara strain in CEF and 15 permanent cell lines. The cells could be grouped into three categories: permissive, semipermissive, and nonpermissive. For MVA, the permissive category consisted of primary CEF, a quail cell line derived from QT6, and the Syrian hamster cell line BHK-21. Only in BHK-21 cells did the virus yield approach that occurring in primary CEF. The semipermissive category included two African green monkey cell lines: BS-C-1 and CV-1. The nonpermissive category for MVA consisted of three human cell lines HeLa, 293, and SW 839; one rhesus monkey cell line FRhK-4; two Chinese hamster cell lines CHO and CHL; one pig cell line PK(15); and three rabbit cell lines RK13, RAB-9, and SIRC. The grouping for MVA with a restored K1L host range gene was similar except for the inclusion of RK13 cells among permissive lines. The grouping for the Ankara strain, however, was quite different with more permissive and semipermissive cell lines. Nevertheless, in cells that were permissive for MVA, the virus replicated to higher levels than Ankara, consistent with both positive and negative growth elements associated with the adaptation of MVA. The cell lines were also characterized according to their susceptibility to MVA-induced cytopathic effects, expression of a late promoter regulated reporter gene by an MVA recombinant, and stage at which virion morphogenesis was blocked. Finally, the permissive BHK-21 cell line was shown to be competent for constructing and propagating recombinant MVA, providing an alternative to primary CEF.