Several attenuated strains of pseudorabies virus contain genomes that carry a deletion in their short unique (Us) component. The sizes of the deletions are different in the various attenuated strains; the deletions may include part of one of the inverted repeats as well as part of the Us region of the genome. In most cases, the deletion includes the gene encoding the glycoprotein gI. The attenuated strains with a deletion in their S component have a common history of having been cultivated in chicken embryo fibroblasts (CEF). We show here that passage of wild-type virus in CEF promotes the emergence of populations of virions with a deletion in their S component. The emergence of these mutants is the result of their growth advantage over the wild type and is related to the lack of expression of gI, as shown by the following. (i) The Norden strain (which has a deletion in the Us) was marker rescued to restore an intact Us. The nonrescued Norden strain had a growth advantage over the rescued Norden strain in CEF. (ii) Passage of wild-type (gI+) virus in CEF but not in rabbit kidney or pig kidney cells resulted invariably in the emergence of virions whose genomes had a deletion in the S component. (iii) Passage of a gI- mutant in CEF did not result in the emergence of such virions. The emergence of virions with a deletion in their S component thus appears to be linked to gI expression. We conclude that gI is deleterious to the growth of pseudorabies virus in CEF and that this effect is cell type specific.