Aims: The aim of this study was to evaluate the TEMPO (R) EB automated most probable number (MPN) system for the enumeration of Enterobacteriaceae in comparison with three standard laboratory methods. Method and Results: Pure cultures were used to determine the sensitivity and specificity of the TEMPO (R) EB test. These studies indicated that Enterobacteriaceae were reliably detected with a detection limit below 10 CFU, whilst non-Enterobacteriaceae strains gave negative results at all inoculum levels tested. A total of 209 food, dairy and environmental samples were tested using the four methods, and results indicated that there was no statistically significant difference between the TEMPO (R) EB and either the MPN, pour plate or spread plate techniques. Conclusions: It is considered that, with a small number of exceptions, TEMPO (R) EB is a suitable method for the analysis of routine food, dairy and environmental samples as it has many advantages, and the results obtained are equivalent to those of standard methods. Significance and Impact of the Study: The TEMPO (R) system could provide significant benefits to Food Water and Environmental Microbiology laboratories, by responding to food and dairy hygiene problems rapidly (up to 4 days sooner than previously), reducing media and consumable costs and making savings in hands-on staff time.