A new method of assessing substrate utilization in gastrointestinal mucosal specimens is described. Small human endoscopic biopsy specimens with wet weights ranging between 1.4 and 12.2 mg were used to quantify the oxidation of three metabolic substrates, glucose, glutamine and butyrate, through to carbon dioxide over a 2-h period. The technique proved to be reproducible and capable of distinguishing variations in mucosal metabolism between individuals (P < 0.0001 for each substrate). Results were similar to those obtained previously using human and rat colonocytes. To characterize the metabolism of the healthy large bowel, specimens were obtained from five regions in 15 patients who had a normal colonoscopic examination. The results show that butyrate is the preferred fuel source of large bowel mucosa, followed by glutamine, then glucose (P < 0.01). There was no significant regional variation in utilization of the three substrates between the five regions; with respect to glutamine, this is contrary to previous findings.