A characterization of genes B13R (SPI-2) and B22R (SPI-1) from vaccinia virus strain Western Reserve (WR) is presented. These genes are transcribed early during infection and the predicted encoded proteins show similarity to the superfamily of serine protease inhibitors (serpins). The 5′ transcriptional initiation site of each gene was mapped by primer extension experiments to 71–72 and 31 nucleotides upstream of the B13R and B22R open reading frames (ORFs), respectively. Each ORF was expressed in Escherichia coli and specific antisera were raised against the protein produced. These antisera were used to identify the B13R- and B22R-encoded proteins in vaccinia virus-infected cells as stable, intracellular, nonglycosylated proteins of M_r 38.5K and M_r 40K, respectively. The B22R gene product was detected in all orthopoxviruses tested including cowpox, rabbitpox, and vaccinia strains WR, Copenhagen, Tashkent, Tian Tan, Lister, Wyeth, IHD-J, and IHD-W. In contrast, the B13R gene product had a more limited distribution and was not detected in Copenhagen, Tashkent, Lister, and Tian Tan. Viable virus deletion mutants that lacked only B13R or B22R coding sequences (ΔB13R and ΔB22R) and revertant viruses in which the deleted gene was restored were constructed by transient dominant selection. The growth of the deletion mutants in cell culture was indistinguishable from that of wild-type virus. Additionally the virulence of each deletion mutant was indistinguishable from wild-type and revertant viruses in a murine intranasal model.