SummaryThe aim of this study was to explore the relationships between a Gram-positive bacterial population and methane production in the rumen of dairy cows supplemented with monensin and tallow, alone or in combination. Under each treatment condition, the following ruminal fermentation parameters were measured: pH, oxidationreduction potential (ORP), VFA, and methane production. Quantification of Gram-positive bacteria present in the rumen was performed by amplifying the 16S rDNA gene using PCR. Diversity analysis of Gram-positive bacteria was performed by PCR-denaturing gradient gel electrophoresis (DGGE), and bacterial taxonomy data were obtained by bioinformatics tools. Neither additive affected the pH or ORP, but they both reduced the total quantity of VFA. Supplementation with monensin, tallow, or their combination caused a decrease in methane production compared to the control diet. The best treatment to reduce methane production was monensin, with a reduction of 7.2% compared to the control diet. Supplementation with monensin or tallow did not affect the bacterial population. The Shannon diversity index was higher with monensin supplementation than with the other treatments. The analysis of each DGGE microbial community by the unweighted pair group method with averaging (UPGMA) revealed two clusters, one group with the control and monensin diets and the other with the tallow and combination diets. Taxonomic analysis of the dominant bands from the DGGE gel with the use of the Ribosomal Database Project (RBP) revealed that the Ruminococcaceae family was predominant, followed by the Lachnospiraceae family. Correspondence analysis (CA) suggested that both were negatively correlated with methane production in all treatments.