Murid herpesvirus 4 (MuHV-4) microRNAs were previously cloned from latently infected tumor cells and predicted to be processed from a series of RNA polymerase III primary transcripts. We detected maturely processed MuHV-4 miRNAs within total RNA from lytically infected cells in vitro and infected tissues ex vivo, using a highly sensitive reverse ligation meditated RT-PCR strategy. We determined that the MuHV-4 microRNAs are biologically active during infection by a luciferase reporter system. We experimentally demonstrated that transcription of the MuHV-4 microRNAs is by RNA polymerase III by alpha-amanitin insensitivity and by specific deletion of the RNA polymerase III type 2-like promoter elements of MuHV-4, resulting in the complete loss of miRNA detection and function. Finally, we demonstrate that these 10 viral miRNAs, each transcribed from highly conserved and novel polymerase III promoter elements, vary markedly in their relative abundance and activity.