Barley, cornstarch, crested wheatgrass, glucose, molasses and solka floc were fermented with rumen fluid obtained from sheep that were adapted or unadapted to feed grade biuret (Kedlor) in their diet. Each rumen fluid and substrate combination was subjected to three nitrogen treatments: (1) control, (2) nitrogen added as feed grade biuret and (3) reagent grade biuret in a factoral arrangement of treatments. In vitro fermentations were terminated at 0, 12, 24, and 36 hours and the protein nitrogen insoluble in trichloroacetic acid (TCA) was determined. Molasses and cornstarch fermentation residues increased while barley, crested wheatgrass, glucose and solka floc decreased in TCA insoluble protein nitrogen. The average of all treatments decreased in TCA insoluble protein nitrogen during the first 12 hours and increased during the remaining 24 hours. Unadapted rumen fluid with simple carbohydrates from glucose and molasses and adapted rumen fluid with complex carbohydrates from barley, crested wheatgrass, cornstarch and solka floc gave the largest gains in TCA insoluble protein nitrogen. No significant differences occurred due to the addition of non-protein nitrogen.