Efforts to characterize the human transcriptome have largely been limited to blood, urine, and tissue analyses (i.e., normally sterile materials). We report here an extraction protocol using commercially available reagents to obtain high-yield, reverse-transcribable RNA from human stool. Quantitative reverse transcriptase polymerase chain reactions demonstrated minimal intra-specimen but considerable intra-subject variability over time of transcripts for interleukin-6 (IL-6), IL-8, epidermal growth factor (EGF), calprotectin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This technique now expands opportunities to use the human fecal transcriptome to characterize gastrointestinal pathophysiology.