Aims: To develop a real-time Taq nuclease assay (TNA) to enable the in vivo enumeration of Megasphaera elsdenii. Methods and Results: Megasphaera elsdenii YE34 was phenotypically characteristic of the species and had 16S rDNA sequence similarity of 980,6 to previously described isolates. Calibration of the number of cells of M. elsdenii against the cycle threshold of fluorescent dye release gave a straight-line relationship with a correlation coefficient approximating unity. The specificity of the assay for M. elsdenii was confirmed by performing it against a panel of 24 heterogenous, mainly ruminal bacteria. Megasphaera elsdenii was not detected in ruminal contents from a pasture-fed steer but was readily detected 2 and 50 h after the probiotic introduction of the bacterium into the rumen. Conclusions: Real-time TNA has provided a sensitive and specific means of enumerating the M. elsdenii population in rumen contents. Significance and Impact of the Study: Megasphaera elsdenii is an important lactate-degrading ruminal bacterium that has been selected for probiotic use to prevent acidosis and enhance starch utilization in grain-fed cattle. The assay developed in this study provides a tool for determining the ability of probiotically-introduced M. elsdenii to establish useful populations in the rumen.