ABSTRACT Excessive NH ₃ production in the rumen is a major nutritional inefficiency in ruminant animals. Experiments were undertaken to compare the rates of NH ₃ production from different substrates in ruminal fluid in vitro and to assess the role of asaccharolytic bacteria in NH ₃ production. Ruminal fluid was taken from four rumen-fistulated sheep receiving a mixed hay-concentrate diet. The calculated rate of NH ₃ production from Trypticase varied from 1.8 to 19.7 nmol mg of protein ⁻¹ min ⁻¹ depending on the substrate, its concentration, and the method used. Monensin (5 μM) inhibited NH ₃ production from proteins, peptides, and amino acids by an average of 28% with substrate at 2 mg/ml, compared to 48% with substrate at 20 mg/ml ( P = 0.011). Of the total bacterial population, 1.4% grew on Trypticase alone, of which 93% was eliminated by 5 μM monensin. Many fewer bacteria (0.002% of the total) grew on amino acids alone. Nineteen isolates capable of growth on Trypticase were obtained from four sheep. 16S ribosomal DNA and traditional identification methods indicated the bacteria fell into six groups. All were sensitive to monensin, and all except one group (group III, similar to Atopobium minutum ), produced NH ₃ at >250 nmol min ⁻¹ mg of protein ⁻¹ , depending on the medium, as determined by a batch culture method. All isolates had exopeptidase activity, but only group III had an apparent dipeptidyl peptidase I activity. Groups I, II, and IV were most closely related to asaccharolytic ruminal and oral Clostridium and Eubacterium spp. Group V comprised one isolate, similar to Desulfomonas piger (formerly Desulfovibrio pigra ). Group VI was 95% similar to Acidaminococcus fermentans. Growth of the Atopobium - and Desulfomonas -like isolates was enhanced by sugars, while growth of groups I, II, and V was significantly depressed by sugars. This study therefore demonstrates that different methodologies and different substrate concentrations provide an explanation for different apparent rates of ruminal NH ₃ production reported in different studies and identifies a diverse range of hyper-ammonia-producing bacteria in the rumen of sheep.