In dairy rations, Met is often a limiting amino acid that is provided by rumen-undegradable protein and rumen-protected sources of Met. A Met precursor, 2-hydroxy-4-(methylthio) butanoic acid (HMB) has undergone considerable study for ruminal and postruminal metabolism, whereas its isopropyl ester (HMBi) has been evaluated primarily with respect to its supply of metabolizable Met rather than as a preformed source of Met for microbial metabolism. A control and 3 isomolar Met treatments-0.097% dl-Met, 0.048% dl-Met plus 0.055% HMBi (Met + HMBi treatment), and 0.11% HMBi-were pulse-dosed every 8h into continuous cultures simultaneously with feeding. Treatment had no effect on digestibilities of acid-detergent fiber or true organic matter. Digestibilities of neutral detergent fiber and hemicellulose were linearly decreased with increasing HMBi inclusion. Concentration of NH3-N tended to decrease linearly and quadratically, and NH3-N flow tended to decrease linearly, with increasing HMBi inclusion; in contrast, the proportion of bacterial N derived from NH3-N increased linearly. Peptide N increased linearly and tended to be affected quadratically (highest for the HMBi treatment). Acetate and propionate production both decreased with increasing HMBi, but acetate declined more such that acetate:propionate increased linearly. Isobutyrate production decreased, but isovalerate and valerate increased with increasing HMBi inclusion. Relative changes in population abundance were not detected by denaturing gradient gel electrophoresis. In the second study, which was done in batch culture, Met treatments consisted of control, 0.097% l-Met, 0.097% l-Met, 0.125% dl-HMBi, 0.098% dl-HMB, 0.250% dl-HMBi (2× HMBi), 0.049% dl-Met + 0.063% dl-HMBi (Met + HMBi), and 0.098% dl-HMB + 0.039% isopropanol. All of these Met treatments were unlabeled (i.e., at natural abundance of (13)C) but simultaneously dosed with equivalent dosages of [1-(13)C]-l-Met. All 8 treatments were inoculated with faunated or partially defaunated inocula. Protozoal abundance had minor effect on measurements. The unlabeled l-Met treatment had the lowest (13)C enrichment of Met in the microbial pellet followed by Met + HMBi and then d-Met or dl-HMB, which were lower than remaining treatments. The percentage of the [1-(13)C]-l-Met dose recovered in microbial Met was lowest for the l-Met treatment; intermediate for d-Met, dl-HMB (with or without isopropanol), and Met + HMBi treatments; and highest for HMBi, 2× HMBi, and control. Results suggest that racemization of d-Met lags behind l-Met. The similar conversions of the HMBi and 2× HMBi treatments compared with the control suggests a low degradation of HMBi to provide unlabeled Met to dilute the [1-(13)C]-l-Met dose for protein synthesis. The lack of treatment by time interaction suggests that these initial responses carried through during the 24h of incubation. The proportion of HMBi available to ruminal microbes can influence microbial metabolism, potentially through formation of l-Met.