Ectromelia virus encodes a protein which is homologous to the product of the vaccinia virus host range gene, K1L, except for eight conservative and two non-conservative substitutions and an additional threonine residue at the carboxyl terminus. Unlike the vaccinia virus gene, the ectromelia virus homolog failed to support optimal virus replication in RK-13 cells and appeared to be expressed 20-fold less efficiently. This lower level of expression was not due to the genetic background of the virus, K1L RNA transcription, sequence of the K1L RNA leader, or stability of K1L RNA or protein. Infections of RK-13 cells with ectromelia or vaccinia virus mutants lacking an intact K1L gene resulted in transient expression of early genes followed by a rapid and irreversible cessation of both virus and host protein synthesis. Infections of the disease-susceptible ANCR or -resistant C57BL/6 mice with the K1L-lacking ectromelia virus yielded a pathogenesis pattern indistinguishable from wild-type, suggesting that the ectromelia virus homolog of vaccinia virus K1L is not important for ectromelia virus in vivo replication and spread.