Epstein-Barr virus (EBV) has been shown to encode at least 40 microRNAs (miRNAs), an important class of molecules that negatively regulate the expression of many genes through post-transcriptional mechanisms. Here we have used real time PCR assays to quantify the levels of EBV-encoded BHRF1 and BART miRNAs in latently infected cells and in cells induced into lytic cycle. During latency, BHRF1 miRNAs were only seen in cells with detectable Cp and/or Wp-initiated EBNA transcripts, while the BART miRNAs were expressed in all forms of latent infection. Surprisingly, levels of different BART miRNAs were found to vary up to 50-fold within a cell line. However this variation could not be explained by differential miRNA turnover as all EBV miRNAs appeared to be remarkably stable. Following entry into virus lytic cycle miR-BHRF1-2 and 1-3 were rapidly induced, coincident with the onset of lytic BHRF1 transcripts, while miR-BHRF1-1 expression was delayed until 48 h and correlated with the appearance of Cp/Wp-initiated EBNA transcripts. In contrast, levels of BART miRNAs were relatively unchanged during virus replication, despite dramatic increases in BART transcription. Finally we show that BHRF1 and BART miRNAs were delayed relative to the induction of BHRF1 and BART transcripts in freshly infected primary B cell cultures. In summary, our data show that changes in BHRF1 and BART transcription are not necessarily reflected in altered miRNA levels, suggesting that miRNA maturation is a key step in regulating steady state levels of EBV miRNAs.