The objective of this study was to compare the effects of slow-release urea (SRU) or free urea on rumen fermentation using an in vitro model. Low energy (20:80, wheat: grass silage) and high-energy rations (60:40, wheat: grass silage) were treated with 3 different nitrogen sources: soybean meal (SBM), urea and a SRU product (Optigen Alltech Inc., KY). SBM replaced 20, 60 or 100 mg of the 1000 mg diets. The dose of urea or SRU was calculated to provide the same amount of nitrogen as the SBM treatments, with the basic diet substituted with 2.3, 6.9 and 11.5 mg for the urea treatments and with 2.6, 7.8, 13.1 mg for the SRU treatments. This design resulted in 20 treatments with 5 replicates each. The components of each treatment were weighed in serum bottles which were flushed with CO₂ and sealed, after which 38 mL of anaerobic, reduced, buffer solution (+38°C) was introduced under oxygen free CO₂ flow. Simulation began when 2 mL of freshly strained rumen fluid was added and continued for 24 h. Ammonia, lactic acid and total gas production were measured. Two-tailed t-tests were used to analyze differences between treatments and controls and between the SRU and urea treatments. The addition of urea significantly increased (P < 0.001) ammonia concentrations in low energy diets after 9 and 12 h, while the level of ammonia did not differ between SRU and control treatments. For high-energy diets urea led to modest increases in ammonia (P ≤ 0.05) after 9 h, with no differences observed between SRU, SBM and control treatments. In terms of lactic acid accumulation, SRU resulted in a 10–39% (P < 0.1) lower accumulation of lactic acid than the urea for the low energy diets, and 21–33% lower (P ≤ 0.01) accumulation for the high-energy diets. Total gas production for the high-energy diets increased significantly (P < 0.001) for both urea and SRU treatments in a dose-dependent manner while the corresponding amount of SBM had no effect. These findings indicate that the use of SRU results in lower ammonia and lactic acid accumulation when compared with free urea.