A simple selection system has been developed for the cloning and expression of open reading frames in vaccinia virus. The selection system is based on a conditional lethal (host range) mutant of vaccinia virus. A deletion mutant of the vaccinia virus WR strain was generated by insertion of the neomycin resistance gene from transposon Tn5 and selection with the antibiotic G418. This deletion recombinant, vP293, lacked approximately 21.7 kilobases of DNA beginning 3.8 kilobases from the left end of the genome, vP293, was capable of plaquing on primary chicken embryo fibroblasts and two monkey cell lines (BSC-40 and Vero) but was defective in replication in the human cell line MRC-5. Insertion of the host range gene K1L into vP293 restored the ability to grow on MRC-5 cells. A series of plasmids were constructed which in addition to the K1L gene contained a vaccinia virus early-late promoter, H6, followed by a unique polylinker sequence, translational initiation and termination signals, and an early transcription termination signal. These plasmids, pHES1 through 4, allowed for rapid single-step cloning and expression of any open reading frame when recombined in vivo with vP293 and scored for growth on MRC-5 cells.