Supplementation of diets for ruminants with methionine analogues such as 2-hydroxy-4-(methylthio) butanoic acid (HMB) and its isopropyl ester (HMBi) positively affect milk composition and yield, potentially partly through ruminal actions. Our objective was to investigate effects of HMB and HMBi on the rumen microbial ecosystem, with special emphasis on fibrolytic activities and fibrolytic microorganisms. Six ruminally cannulated Holstein dry cows were randomly assigned to three treatments in a 3 x 3 Latin square design with two cows per cell. Cows were fed twice a day at 7.5 kg/d dry matter (DM) intake with a control diet based on hay and wheat (50150 on a DM basis) supplemented or not with HMB (Rhodimet (R) AT88, Adisseo at 1.25 g/kg DM intake) or HMBi (MetaSmart (TM), Adisseo, at 2.50 g/kg DM intake). Substrate degradability (i.e., corn grain, corn silage) was evaluated by in sacco incubation and effective degradability measurements of DM, crude protein, starch and neutral detergent fibre. Fermentation products (i.e., volatile fatty acids (VFAs), ammonia, lactate) and fibrolytic enzyme activities (i.e., carboxymethylcellulase (CMCase), xylanase) were analyzed in rumen content samples before (-1 h) and after (+3 h) feeding. Protozoal populations were counted by microscopy. Abundances in total and fibrolytic bacteria Fibrobacter succinogenes, Ruminococcus flavefaciens and Ruminococcus albus in total rumen contents or adhering to in sacco residues were estimated using quantitative polymerase-chain reaction (qPCR). HMB and HMBi supplementation increased VFA concentrations in the rumen as well as the ruminal abundance of F. succinogenes (P=0.03) and R. flavefaciens (P=0.05), although these increases had no effect on the CMCase and xylanase activities of the rumen contents, nor on in sacco bacterial colonization and degradation of the two corn substrates. Under our experimental conditions, Met analogues enhanced growth of two cellulolytic bacterial representatives, but did not improve rumen fibrolytic activity. (C) 2013 Elsevier B.V. All rights reserved.