Technical Abstract: Mixed ruminal microbes were incubated for 24 h in vitro with mixed forage and concentrate rations containing 20% or 30% non-fiber carbohydrates (NFC) to assess in vitro fiber digestibility, fermentation end products, and relative population sizes (RPS, expressed as a percentage of 16S rRNA gene copies determined by quantitative real-time PCR) of various bacterial taxa. Several taxa displayed substantial decreases in RPS (genus Prevotella, three Prevotella species, Rumincoccus flavefaciens, Eubacterium ruminantium, and Butyrivibrio fibrisolvens), while two (Rumincocccus albus and Megasphaera elsdenii) displayed substantial increases. RPS of 7 other taxa were not changed after the same incubation period. Among incubations containing 30% NFC, shifts in RPS were remarkably independent of fermentation conditions (initial pH 6.2 vs. 6.8, or 0% vs 1% corn oil). Similar in vitro experiments with the same ration in the presence of monensin, and using inocula from the same cows following adaptation to monensin, revealed RPS patterns generally similar to those of unadapted inocula without monensin. Within the range of conditions tested, monensin improved in vitro fiber digestibility regardless of environmental conditions, but significant changes in fermentation end-product formation were only observed at the higher NFC level in combination with the lower pH, and this effect was enhanced by the presence of corn oil. The data indicate that in vitro conditions can substantially change the quantitative distribution of microbial populations, and that these changes are both inherent in the in vitro method, and distinct from specific differences in divergent in vitro environmental conditions.