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Creating a balanced amino acid (AA) profile for rumen microbes increases efficiency of feed conversion into microbial protein , which decreases manure N. Methionine (MET) is a limiting AA in lactating dairy cows. A steady supply of MET reduces the need for rumen bacteria to synthesize AA from carbon skeletons. A previous study conducted in the OSU dairy lab evaluated the supplementation of 2-hydroxy-4-(methylthio) butanoic acid (HMB) isopropyl ester (HMBi; 0.11%), MET (0.097%), HMBi + MET (0.055% + 0.048%) and control (CON) to continuous culture (CC) fermenters. Extracellular MET pools were higher for HMBi treatments than MET, but microbial protein synthesis (MPS) did not differ. We hypothesized MET accumulation may be due to differences in bacterial utilization of MET stereoisomers, because isopropanol (ISO; hydrolyzed from HMBi) may affect membrane fluidity and HMBi is more slowly converted to MET. This expanded treatments (TRT) to CON (1), L-MET (2; 0.097%), D-MET (3; 0.097%), HMBi (4; 0.125%), HMB (5; 0.098%), 2×HMBi (6; 0.250%), ½HMBi + ½DL-MET (7; 0.063% + 0.049%), and HMB + ISO (8; 0.098% + 0.039%). We investigated the effects of the TRTs with batch culture (BC) fermentation. Protozoa were washed out of fermenters in the CC study but traditional BC inocula contain protozoa. Therefore, rumen fluid was collected from 2 Holstein cows and CC was used to prepare faunated (F) and defaunated (D) inocula for use in BC. In addition to TRTs, BC tubes were dosed with 15N enriched (NH4)2SO4 to compare incorporation of 15NH3 into MICROBIAL PROTEIN. Each TRT contained 6 replicates (D and F inocula each dosed to 3 replicates) with measurements taken at 0, 2, 8, and 24 h. Measurements included TCA soluble N (SN), 15NH3, NH3, peptides (PEP; SN–NH3), 15N enriched non-ammonia-N (NAN), and total NAN. SN, NH3, and PEP data were analyzed using the mixed procedure of SAS to determine main effects and interactions (other data in progress). The main effect of time was significant for SN, NH3, and PEP; inocula for SN (D=23.38, F=25.05 mg/dL) and NH3 (D=10.50, F=12.28 mg/dL); no other main effects were found. Interactions were found (data not shown) for inocula×TRT×time for NH3 (2h) and PEP (8, 24h), inocula×time for SN (2, 8h) and NH3 (2, 8h), and TRT×time for PEP (8, 24h). These data indicate possible differences in MET utilization but data completion will allow for more specific conclusions. Application of this data may improve feed efficiency of rumen microbes and dairy cows, which could reduce feed costs and N pollution.