During latent infections of sensory neurons, herpes simplex virus type 1 gene expression is restricted to the latency-associated transcripts (LATs). The association of the stable 2.0-kb LAT intron with polysomes has suggested that it might represent a novel mRNA. In this work, we investigated expression of 2.0-kb LAT open reading frames (ORFs) by inserting the gene for green fluorescent protein (GFP) within the 2.0-kb LAT sequence, both within a LAT expression plasmid and in the context of the virus. Upon transient transfection of cells of both neuronal and nonneuronal origin with LAT-GFP expression vectors, low-level GFP fluorescence was distributed over the cell cytoplasm and likely resulted from infrequent initiation at a GFP AUG codon, on either unspliced or alternately spliced LAT RNAs. A second nucleolar GFP expression pattern which resulted from fusion of GFP to a conserved ORF in exon 1 of the LAT gene was also observed. However, the abundant expression of this fusion protein was dependent upon an artificially added translation initiation codon. Expression was much reduced and restricted to a small subset of transfected cells when this initiator codon was removed. Neither the 2.0-kb LAT-GFP intron itself nor transcripts originating from the latency-associated promoter 2 (LAP2) were responsible for GFP expression. Abundant alternate splicing involving the 1.5-kb LAT splice acceptor and including splicing between the 1.5-kb LAT splice donor and acceptor, was observed in the nonneuronal Cos-1 cell line. Contrary to the results of our transfection studies, GFP expression could not be detected from a LAT-GFP virus at any stage of the infection cycle. Our results suggest that the inhibition of LAT ORF expression during viral infection occurred primarily at the level of translation.