The glycoprotein gII, one of the major glycoproteins of pseudorabies virus (PRV), is represented by a complex of three related glycopolypeptides. There is evidence that two of them, linked by disulfide bonds, arise by proteolytic cleavage of the larger precursor glycoprotein. Using specific antisera and a monoclonal antibody against the glycoprotein complex one single nonglycosylated in vitro translated precursor polypeptide with mol wt 110,000 was identified. Mapping of the gene coding for this polypeptide was achieved by hybrid selection of late viral RNA on cloned DNA fragments. The structural gene for the gII complex was found to reside in the long unique part of the PRV genome on BamHI fragment 1 and SalI subfragments 1A and G (map units 0.105 to 0.130). A 3.5-kb mRNA was identified as the probable gII-specific transcript. In addition, further polypeptides encoded in the BamHI fragment 1 were described and RNAs were characterized by Northern blot hybridizations with the cloned SalI subfragments.