ABSTRACT The in vivo production of nitrous oxide (N ₂ O) by earthworms is due to their gut microbiota, and it is hypothesized that the microenvironment of the gut activates ingested N ₂ O-producing soil bacteria. In situ measurement of N ₂ O and O ₂ with microsensors demonstrated that the earthworm gut is anoxic and the site of N ₂ O production. The gut had a pH of 6.9 and an average water content of approximately 50%. The water content within the gut decreased from the anterior end to the posterior end. In contrast, the concentration of N ₂ O increased from the anterior end to the mid-gut region and then decreased along the posterior part of the gut. Compared to the soil in which worms lived and fed, the gut of the earthworm was highly enriched in total carbon, organic carbon, and total nitrogen and had a C/N ratio of 7 (compared to a C/N ratio of 12 in soil). The aqueous phase of gut contents contained up to 80 mM glucose and numerous compounds that were indicative of anaerobic metabolism, including up to 9 mM formate, 8 mM acetate, 3 mM lactate, and 2 mM succinate. Compared to the soil contents, nitrite and ammonium were enriched in the gut up to 10- and 100-fold, respectively. The production of N ₂ O by soil was induced when the gut environment was simulated in anoxic microcosms for 24 h (the approximate time for passage of soil through the earthworm). Anoxia, high osmolarity, nitrite, and nitrate were the dominant factors that stimulated the production of N ₂ O. Supplemental organic carbon had a very minimal stimulatory effect on the production of N ₂ O, and addition of buffer or ammonium had essentially no effect on the initial N ₂ O production rates. However, a combination of supplements yielded rates greater than that obtained mathematically for single supplements, suggesting that the maximum rates observed were due to synergistic effects of supplements. Collectively, these results indicate that the special microenvironment of the earthworm gut is ideally suited for N ₂ O-producing bacteria and support the hypothesis that the in situ conditions of the earthworm gut activate ingested N ₂ O-producing soil bacteria during gut passage.