The availability of viral genes in cloned DNA forms, in addition to the parallel development of an efficient cell-free system capable of producing viral proteins in vitro, has provided new opportunities for the identification of important T cell Ag encoded within viral genomes. We have cloned bovine herpesvirus type 1 surface glycoproteins gI, gIII, and gIV into the RNA transcription vector pE5LVPO. After translation of the resulting viral mRNA in rabbit reticulocyte lysates, each cloned viral protein specifically stimulated bovine T lymphocytes previously primed to BHV-1. In addition, a panel of specifically truncated peptides has allowed the identification of at least two T lymphocyte epitopes encoded within gIII of BHV-1. Our results represent a novel approach for viral Ag synthesis and subsequent detection of lymphocyte recognition. These findings will likely have important implications in the mapping of T lymphocyte epitopes.