Relative quantification real-time PCR was used to quantify several bacterial species in ruminal samples from two lactating cows, each sampled 3 h after feeding on two successive days. Abundance of each target taxon was calculated as a fraction of the total 16S rRNA gene copies in the samples, using taxon-specific and eubacterial domain-level primers. Bacterial populations showed a clear predominance of members of the genus Prevotella, which comprised 42% to 60% of the bacterial rRNA gene copies in the samples. However, only 2% to 4% of the bacterial rRNA gene copies were represented by the classical ruminal Prevotella species Prevotella bryantii, Prevotella ruminicola and Prevotella brevis. The proportion of rRNA gene copies attributable to Fibrobacter succinogenes, Ruminococcus flavefaciens, Selenomonas ruminantium and Succinivibrio dextrinosolvens were each generally in the 0.5% to 1% range. Proportions for Ruminobacter amylophilus and Eubacterium ruminantium were lower (0.1% to 0.2%), while Butyrivibrio fibrisolvens, Streptococcus bovis, Ruminococcus albus and Megasphaera elsdenii were even less abundant, each comprising < 0.03% of the bacterial rRNA gene copies. The data suggest that the aggregate abundance of the most intensively studied ruminal bacterial species is relatively low and that a large fraction of the uncultured population represents a single bacterial genus.