Aims: The aim of this study was to develop and optimize a novel method that combines ethidium bromide monoazide (EMA) staining with real-time PCR for the detection of viable Escherichia coli O157:H7 in ground beef. EMA can penetrate dead cells and bind to intracellular DNA, preventing its amplification via PCR. Methods and Results: Samples were stained with EMA for 5 min, iced for 1 min and exposed to bright visible light for 10 min prior to DNA extraction, to allow EMA binding of the DNA from dead cells. DNA was then extracted and amplified by TaqMan (R) real-time PCR to detect only viable E. coli O157:H7 cells. The primers and TaqMan (R) probe used in this study target the uidA gene in E. coli O157:H7. An internal amplification control (IAC), consisting of 0 center dot 25 pg of plasmid pUC19, was added in each reaction to prevent the occurrence of false-negative results. Results showed a reproducible application of this technique to detect viable cells in both broth culture and ground beef. EMA, at a final concentration of 10 mu g ml-1, was demonstrated to effectively bind DNA from 108 CFU ml-1 dead cells, and the optimized method could detect as low as 104 CFU g-1 of viable E. coli O157:H7 cells in ground beef without interference from 108 CFU g-1 of dead cells. Conclusions: EMA real-time PCR with IAC can effectively separate dead cells from viable E. coli O157:H7 and prevent amplification of DNA in the dead cells. Significance and Impact of the Study: The EMA real-time PCR has the potential to be a highly sensitive quantitative detection technique to assess the contamination of viable E. coli O157:H7 in ground beef and other meat or food products.