The IR6 protein of different plaque isolates from three passages of the equine herpesvirus 1 strain Rac was investigated. Southern blot and DNA sequence analyses revealed that plaque isolates from the 12th passage (RacL11 and RacL22) retained both copies of the IR6 gene, whereas two different genotypes were observed by the 185th passage: RacM24 still harbored both copies of the IR6 gene, whereas RacM36 deleted one of the two copies. In the 256th passage (RacH), both copies of the IR6 gene were absent. As compared to the wild-type IR6 protein, both the RacM24 and RacM36 IR6 protein displayed amino acid exchanges at positions 34, 42, 110, and 134 of the 272 amino acid polypeptide. It is shown that (i) the IR6 protein is nonessential for virus growth in vitro. (ii) In RacL11-infected equine and rodent cells, the typical rod-like appearance of the IR6 protein could be detected from 6 hr p.i., whereas in RacM24- and RacM36-infected cells formation of these structures was not observed. (iii) The RacL11 IR6 gene product was present in both the nuclei and cytoplasmic fraction of infected cells. In contrast, the IR6 protein of both RacM24 and RacM36 colocalized with cytoplasmic membrane vesicles. (iv) The RacL11 and RacL22 IR6 protein is present in viral nucleocapsids, whereas that of RacM24 and RacM36 is not incorporated into virions. (v) The RacL11 IR6 gene product aggregated to disulfide-linked oligomers, whereas the RacM24 and RacM36 IR6 protein showed only marginal oligomerization. (vi) In COS7 cells transfected with constructs expressing either the full-length RacL11-IR6 protein or a truncated form lacking the 81 carboxyterminal amino acids, the formation of rod-like structures was observed, indicating that another viral protein is not necessary for aggregation of the IR6 protein. In contrast, the IR6 protein expressed from constructs derived from either RacM24 or RacM36 failed to form these structures. (vii) Analyses of chimeric RacL11-RacM24 IR6 proteins suggest a crucial role for amino acid Leu-134 in the ability of the IR6 protein to form the rod-like structures.