Vaccinia virus (VV) K1L is a host-range gene and encodes a protein comprised of six ankyrin repeats (ANKs). We showed here that a large portion of the K1L protein, except ankyrin repeat 1 (ANK1) and C-terminal halves of ANK2 and ANK3, can be deleted or substituted with an unrelated ANK with no adverse effect on VV replication in human HeLa cells. In contrast, only ANK4 and ANK6 can be mutated without impairing VV replication in rabbit RK13 cells. The growth rate of VV in HeLa cells was reduced differentially by substituting phenylalanine 82 or serine 83 of ANK2 and abolished completely by substituting both residues. These substitutions, however, did not affect K1L's ability to bind ACAP2, a GTPase-activating protein for ARF6. Our data support the hypothesis that surface residues of a few consecutive K1L ANKs mediate the host-range function by interacting with protein factors that are distinct from ACAP2.