When ruminal fluid from a cow fed timothy hay was serially diluted (10-fold increments into anaerobic broth containing 15 mg ml(-1) Trypticase), the low dilutions (less than or equal to 10(-6)) had optical densities greater than 2.0 and ammonia concentrations greater than 100 mm. The optical densities and ammonia concentrations of the 10(-8) and 10(-9) dilutions were very low, but large cocci were observed in the 10-8 dilution. The large cocci were isolated and identified by 16S rDNA sequencing as Megasphaera elsdenii. The freshly isolated strain (JL1) grew well on Trypticase, but less than 4% of the amino acid nitrogen in Trypticase was converted to ammonia. Optical density and ammonia production were twice as great if Casamino acids were provided. and similar results were obtained with seven other strains (B159, AW106, YT91, LC1, T81, J1, and YZ70). Specific activities of deamination (based on Casamino acids) of the eight strains ranged from 100 (strain JL1) to 325 (strain B 159) nmol mg protein(-1) min(-1). None of the strains could utilize branched-chain amino acids as an energy source for growth, but specific activities of branched-chain amino acid deamination ranged from 15 to 65 nmol mg protein(-1) min(-1). All eight of the M. elsdenii strains grew well in the presence of 5 muM monensin. and only two of the strains were strongly inhibited by 20 muM monensin. On the basis of these results. it appears that M. elsdenii is deficient in peptidase activity and can utilize only a few amino acids. Some M. elsdenii strains produced ammonia and branched-chain volatile fatty acids nearly as fast as obligate amino acid-fermenting ruminal bacteria, but the extent of this production was at least fourfold tower. Because all of the strains could tolerate 5 muM monensin, it is unlikely that this feed additive would significantly inhibit M. elsdenii in vivo.