Gene expression analyses including RT-PCR, microarrays and metatranscriptomics are techniques that could significantly expand our understanding of the rumen microbial ecosystem. The ability to isolate and stabilize representative RNA samples is critical to obtaining reliable results in all of these procedures. In this study, we established an improved RNA isolation method for extracting high quality total RNA from both liquid and solid phases of ruminal contents. This method is based on liquid nitrogen-mortar disruption and acid guanidinium-phenol-chloroform extraction combined with column purification. Yield of total RNA using this procedure was as high as 150 µg per gram of ruminal content. The typical large subunit/small subunit (LS/SS) rRNA ratio ranged from 1.8 to 2.0 with an RNA integrity number (Agilent) greater than 8.5. The rRNA profile associated with solid ruminal contents was more complex than that associated with the fluid, exhibiting broader rRNA peaks with discrete shoulders. This result is consistent with the isolation of both prokaryotic and eukaryotic rRNA from the particle-associated microbial consortia. The addition of RNAprotect reagent (Qiagen) to the samples resulted in partially degraded RNA, with LS/SS rRNA ratio lower than 1.0, and noticeable smearing of the RNA bands upon electrophoresis. We therefore recommend that this reagent not be used for the isolation of RNA from rumen samples. Our research team is currently applying this new technique to obtain high quality ruminal RNA to characterize the rumen microbiome on a metatranscriptomics level, using next generation sequencing.