An extraction method was developed to recover high-quality RNA from rumen digesta and mouse feces for phylogenetic analysis of metabolically active members of the gut microbial community. Four extraction methods were tested on different amounts of the same samples and compared for efficiency of recovery and purity of RNA. Trizol extraction after bead beating produced a higher quantity and quality of RNA than a similar method using phenol/chloroform. Dissociation solution produced a 1.5- to 2-fold increase in RNA recovery compared with phosphate-buffered saline during the dissociation of microorganisms from rumen digesta or fecal particles. The identity of metabolically active bacteria in the samples was analyzed by sequencing 87 amplicons produced using bacteria-specific 16S rDNA primers, with cDNA synthesized from the extracted RNA as the template. Amplicons representing the major phyla encountered in the rumen (Firmicutes, 43.7%; Proteobacteria, 28.7%; Bacteroidetes, 25.3%; Spirochea, 1.1%, and Synergistes, 1.1%) were recovered, showing that development of the RNA extraction method enables RNA-based analysis of metabolically active bacterial groups from the rumen and other environments. Interestingly, in rumen samples, about 30% of the sequenced random 16S rRNA amplicons were related to the Proteobacteria, providing the first evidence that this group may have greater importance in rumen metabolism than previously attributed by DNA-based analysis.