Iso-α acids from hops (Humulus lupulus) derived from the brewing industry have been shown to exhibit bacteriostatic properties against gram-positive bacteria. Previous research using whole or ground hops has shown promising results for decreasing hyper-ammonia producing bacteria in the rumen. However, hops contain additional fermentable substrate and other metabolites including tannins, β-acids and xanthohumol that confound the direct effects of iso-α-acids on rumen fermentation. Research using strictly iso-α acids in rumen culture is limited. The objective of this study was to examine the direct effects of iso-α acids on fermentation by rumen microbes using a dual-flow continuous culture system. Eight fermenters were used in 2 consecutive 10-d periods consisting of 7 d of adaptation followed by 3 d of sampling. Fermenters were provided with a basal diet consisting of 44% corn silage, 14% alfalfa hay, 13% ground corn, 11% protein mix, 10% corn gluten feed, 5% cottonseed and 3% liquid vitamin and mineral supplements on a DM basis. This diet provided substrate for ruminal microbes maintained in continuous culture at a rate of 75 g of DM/L of fermenter volume/day. Iso-α Extract (IE) solution was added to the artificial saliva buffer to supply 0 (CON), 600 (LOW), 1200 (MED) and 1800 (HIGH) mg of IE/kg of diet DM/day. There was no effect (P > 0.05) on DM, OM, NDF or ADF digestion (%). Volatile fatty acid (VFA) metabolism was not affected by IE treatment (P > 0.05), with total VFA concentrations of 105.5, 93.4, 87.9 and 103.6 mM for the CON, LOW, MED and HIGH treatments, respectively. Similarly, N metabolism was not affected (P > 0.05) by IE level, with the CON, LOW MED, and HIGH treatments resulting in nitrogen concentrations of 7.4, 5.3, 7.6 and 6.8 mg N/dL of rumen fluid, respectively. No significant effects (P > 0.05) on fermenter pH were observed. In conclusion, administration of IE had no effects on measurements of fermentation by ruminal microbes maintained in continuous culture fermenters.