Vaccinia virus gene D12L, which lies between nucleotides 14,350 and 13,487 in the HindIII D fragment, is transcribed at early times in infection and is capable of encoding a protein 287 amino acids in length with a predicted molecular mass of 33,331. A polyclonal antiserum was raised in rabbits to a fusion protein containing 279 amino acids of the D12L protein, and this serum was used to investigate both the time of synthesis and the function of the D12L protein. A combination of Western blot analysis and immunoprecipitation from pulse-labeled and pulse-chased cell extracts demonstrated that the synthesis of a 31-kDa protein begins early in infection, that it reaches a plateau by about 4 hr, and that it is stable in the infected cell. The D12L protein was localized by Western blot analysis of detergent-solubilized virions to the sodium deoxycholate soluble fraction which suggested that it may be a virion core-associated enzyme. Due to the similarity in apparent molecular weight between the D12L protein and the small subunit of the vaccinia mRNA capping complex the anti-D12L antiserum was employed in Western blot analysis of fractions generated during the purification of the virion mRNA capping enzyme. The 31-kDa D12L protein copurified with the virus capping enzyme through chromatography on heparin-agarose and phosphocellulose and also cosedimented with the capping enzyme through a glycerol density gradient. In addition, the anti-D12L antiserum coprecipitated the large subunit of the capping enzyme, confirming that gene D12L encodes the small subunit of the viral mRNA capping enzyme. An insertion mutation which destroys the gene D12L coding sequence was constructed in a plasmid containing a portion of both genes D11L and D12L and this plasmid was used to rescue a ts mutation, in a single step, in the adjacent gene D11L. Southern blot analysis of the re-plaque-purified virus permitted the identification of the mutant virus only when the mutant was propagated in the presence of wild-type helper virus. We concluded from these data that gene D12L is essential for virus propagation in tissue culture.