Fermentation of neutral detergent solubles (NDS) was assessed using a 3 x 3 x 3 factorial arrangement. Three forage species (alfalfa, bromegrass, and orchardgrass) were collected at three maturities and preserved either by freeze drying, oven drying at 50 degrees C, or by ensiling. Each feed sample and its isolated NDF were fermented in vitro and gas production was monitored. Gas yield from NDS was determined as the difference between gas from the unfractionated forage and from its respective NDF. The forages ranged from 23 (immature alfalfa) to 68% NDF (mature orchardgrass). The silages were well fermented with a final pH of < or = 4.5. Increasing maturity decreased the final gas volume but did not change the rate of gas production from the NDS fraction. There was little difference in gas production between freeze-dried and oven-dried forage samples. Ensiling decreased gas yield from the unfractionated forage. The rate of gas production from the NDS fraction of the ensiled forages decreased an average of .05 h-1 compared with the freeze-dried sample. Gas yield from the NDS fraction decreased (from the freeze-dried sample) between 7 and 36% upon ensiling. The curve substraction approach can be used to evaluate the effects of ensiling on the neutral detergent-soluble fraction of forages.