Research over the last few years has demonstrated the increasing role of microRNAs (miRNAs) as major regulators of gene expression in diverse cellular processes and diseases. Several viruses, particularly herpesviruses, also use the miRNA pathway of gene regulation by encoding their own miRNAs. Marek's disease (MD) is a widespread lymphomatous neoplastic disease of poultry caused by the highly contagious Marek's disease virus type 1 (MDV-1). Recent studies using virus-infected chicken embryo fibroblasts have identified at least eight miRNAs that map to the R(L)/R(S) region of the MDV genome. Since MDV is a lymphotropic virus that induces T-cell lymphomas, analysis of the miRNA profile in T-cell lymphoma would be more relevant for examining their role in oncogenesis. We determined the viral and host miRNAs expressed in MSB-1, a lymphoblastoid cell line established from an MDV-induced lymphoma of the spleen. In this paper, we report the identification of 13 MDV-1-encoded miRNAs (12 by direct cloning and 1 by Northern blotting) from MSB-1 cells. These miRNAs, five of which are novel MDV-1 miRNAs, map to the Meq and latency-associated transcript regions of the MDV genome. Furthermore, we show that miRNAs encoded by MDV-1 and the coinfected MDV-2 accounted for >60% of the 5,099 sequences of the MSB-1 "miRNAome." Several chicken miRNAs, some of which are known to be associated with cancer, were also cloned from MSB-1 cells. High levels of expression of MDV-1-encoded miRNAs and potentially oncogenic host miRNAs suggest that miRNAs may have major roles in MDV pathogenesis and neoplastic transformation.