The 130,000 mol wt glycoprotein I (gI) derived from two approx 80-kDa precursors is one of the major constituents of the envelope of pseudorabies virus (PRV) strain Phylaxia. Recently, gI has been shown to be nonessential for PRV replication since several PRV vaccine strains with deletions in the region of the genome encoding the gI gene have been described. In this paper we demonstrate that other alterations affecting gI expression can occur. We describe a PRV field isolate which expresses a single gI precursor molecule pgI of 64,000 mol wt. This precursor is processed into 60,000 mol wt gI. In contrast to PRV Phylaxia, the gI-expressing isolate is not neutralized by anti-gI monoclonal antibodies. Virions expressing the pgI also emerged after serial in vitro passages of the wild-type PRV strain NIA-5 which initially expressed wild-type pgI. Concomitant with the appearance of pgI the pgI disappeared and the resistance of the virus population to neutralization by anti-gI monoclonal antibodies increased. Furthermore, the amount of expression of gI and pgI in single plaque isolates of the PRV strain Ka was found to be highly variable among different plaque isolates and correlated with a different susceptibility to neutralization by anti-gI monoclonal antibodies. In single plaque isolates of strain Phylaxia, however, gI expression appeared to be stable. In all cases, no genomic or transcriptional alterations could be observed. Thus, viruses resistant to anti-gI antibodies occur spontaneously in vivo and in vitro, which argues against the use of gI as a subunit vaccine.