The program(s) of gene expression operating during murine gammaherpesvirus 68 (gammaHV68) latency is undefined, as is the relationship between gammaHV68 latency and latency of primate gammaherpesviruses. We used a nested reverse transcriptase PCR strategy (sensitive to approximately one copy of gammaHV68 genome for each genomic region tested) to screen for the presence of viral transcripts in latently infected mice. Based on the positions of known latency-associated genes in other gammaherpesviruses, we screened for the presence of transcripts corresponding to 11 open reading frames (ORFs) in the gammaHV68 genome in RNA from spleens and peritoneal cells of latently infected B-cell-deficient (MuMT) mice which have been shown contain high levels of reactivable latent gammaHV68 (K. E. Weck, M. L. Barkon, L. I. Yoo, S. H. Speck, and H. W. Virgin, J. Virol. 70:6775-6780, 1996). To control for the possible presence of viral lytic activity, we determined that RNA from latently infected peritoneal and spleen cells contained few or no detectable transcripts corresponding to seven ORFs known to encode viral gene products associated with lytic replication. However, we did detect low-level expression of transcripts arising from the region of gene 50 (encoding the putative homolog of the Epstein-Barr virus BRLF1 transactivator) in peritoneal but not spleen cells. Latently infected peritoneal cells consistently scored for expression of RNA derived from 4 of the 11 candidate latency-associated ORFs examined, including the regions of ORF M2, ORF M11 (encoding v-bcl-2), gene 73 (a homolog of the Kaposi's sarcoma-associated herpesvirus [human herpesvirus 8] gene encoding latency-associated nuclear antigen), and gene 74 (encoding a G-protein coupled receptor homolog, v-GCR). Latently infected spleen cells consistently scored positive for RNA derived from 3 of the 11 candidate latency-associated ORFs examined, including ORF M2, ORF M3, and ORF M9. To further characterize transcription of these candidate latency-associated ORFs, we examined their transcription in lytically infected fibroblasts by Northern analysis. We detected abundant transcription from regions of the genome containing ORF M3 and ORF M9, as well as the known lytic-cycle genes. However, transcription of ORF M2, ORF M11, gene 73, and gene 74 was barely detectable in lytically infected fibroblasts, consistent with a role of these viral genes during latent infection. We conclude that (i) we have identified several candidate latency genes of murine gammaHV68, (ii) expression of genes during latency may be different in different organs, consistent with multiple latency programs and/or multiple cellular sites of latency, and (iii) regions of the viral genome (v-bcl-2 gene, v-GCR gene, and gene 73) are transcribed during latency with both gammaHV68 and primate gammaherpesviruses. The implications of these findings for replacing previous operational definitions of gammaHV68 latency with a molecular definition are discussed.