Real-time polymerase chain reaction (PCR) was used to quantify 16 procaryotic taxa in the rumina of two lactating dairy cows following supply and subsequent withdrawal of the feed additive monensin (13.9 mg/kg of diet dry matter) in a high-starch, silage-based ration. PCR was conducted on DNA from rumen samples collected 6 h post feeding on two successive days before monensin supplementation, after 30 days of monensin supplementation, and at six weekly intervals after monensin withdrawal. Mean values of relative population size (RPS, the percent of bacterial 16S rRNA copy number) for genus Prevotella increased (P < 0.05) from 41.8% without monensin to 49.2% with monensin and declined to 42.5% after monensin withdrawal. Mean RPS values for two biohydrogenating species (Megasphaera elsdenii and Butyrivibrio fibrisolvens) were low (<0.4%) and declined several-fold in response to monensin. Mean RPS values for the biohydrogenating species Eubacterium ruminantium, four cellulolytic species, four starch- or dextrin-fermenting species, and Domain Archaea were not altered (P > 0.10) upon monensin feeding or withdrawal. The data suggest that monensin in high-starch diets does not suppress populations of classical ruminal Gram-positive bacteria or the availability of H2, though it may affect bacteria involved in biohydrogenation of lipids that regulate bovine mammary lipogenesis.