During vaccinia virus (VV) assembly a major polypeptide migrating with an apparent MW of 35K, designated Ag35, is expressed as an early function and becomes an integral component of the lipoprotein envelope surrounding the mature virion. In a previous study evaluating humoral immunity to VV, a prominent response against Ag35 was invariably detected in immunized mice. In the context of our continuing investigations of the structure and function of the vaccinia envelope, with a view to alteration in antigenicity of this agent when used as a vaccine vector for foreign antigens, we carried out detailed mapping of the Ag35 gene, as well as determination of the nucleotide sequence. Use of hybridization-arrested translation, coupled with immunoprecipitation, located this gene within a 2.7-kbp EcoRI fragment of the larger 8.7-kbp HindIII H fragment. By means of S1 endonuclease resistance analysis a viral transcript was identified at the site of the Ag35 gene, where the occurrence of an open reading frame (ORF), corresponding to the transcript, was deduced from DNA sequence determination. However, the ORF encodes a polypeptide of only 22,300 Da predicted MW, which is much lower than the apparent MW estimated from SDS-polyacrylamide gel electrophoresis. The size discrepancy is not due to glycosylation or phosphorylation of Ag35 but may result from a proline-rich sequence which occurs in this polypeptide. To confirm that the ORF recognized in this study does, indeed, encode Ag35, the gene was expressed as a beta-galactosidase fusion protein in pUC19; Escherichia coli transformed with the relevant clones expressed a polypeptide of the appropriate molecular weight and antigenicity, when tested by Western blots. Regarding secondary structure and hydropathicity it can be predicted from the DNA sequence that Ag35 is highly hydrophilic but contains a hydrophobic region at the carboxy terminus, perhaps providing the stretch involved in membrane insertion. Computer search of a bank of protein sequences revealed an unusually strong similarity of 68% between the Ag35 at amino acid positions 44-121 and the G glycoprotein of respiratory syncytial virus at positions 189-264.