It has been demonstrated and confirmed that certain nonsteroidal anti-inflammatory drugs which inhibit cyclooxygenase and the synthesis of prostaglandins and other eicosanoids, can reduce the formation of both colon polyps and cancers in experimental animals given known carcinogens. Additionally, the results of several epidemiologic studies have suggested that nonsteroidal anti-inflammatory drugs may reduce the risk of colon polyp occurrence and/or colon cancer mortality. We have carried out a study to evaluate the methodology of the measurement of prostaglandin E2 (PGE2) in human colonic mucosa because its concentration may serve as a valuable intermediate marker of the pharmacological activity in Phase II studies of nonsteroidal anti-inflammatory drugs as colon cancer preventive agents. We studied all aspects of the actual measurement of PGE2 including the extraction efficiency of the PGE2 from the mucosa, the precision of the assay and calculation of the PGE2 content in terms of milligrams of protein in the sample, the inhibition of PGE2 by indomethacin over time, the reproducibility of the measurement within one homogenate, the rate of PGE2 production over time, the effect of adding indomethacin versus snap freezing on PGE2 production, the stability of PGE2 in tissues over time stored in liquid nitrogen, and the variability of the measurement of PGE2 in separate biopsies from one individual. Our studies indicated that the most reliable method for accurate and consistent measurements of PGE2 was to add the mucosal tissue instantly after biopsy to an indomethacin buffer that effectively inhibited the in vitro formation of PGE2.