The main objectives of this study were (i) to determine if gut wall-associated microorganisms are responsible for the capacity of earthworms to emit nitrous oxide (N2O) and (ii) to characterize the N2O-producing bacteria of the earthworm gut. The production of N2O in the gut of garden soil earthworms (Aporrectodea caliginosa) was mostly associated with the gut contents rather than the gut wall. Under anoxic conditions, nitrite and N2O were transient products when supplemental nitrate was reduced to N2 by gut content homogenates. In contrast, nitrite and N2O were essentially not produced by nitrate-supplemented soil homogenates. The most probable numbers of fermentative anaerobes and microbes that used nitrate as a terminal electron acceptor were approximately 2 orders of magnitude higher in the earthworm gut than in the soil from which the earthworms originated. The fermentative anaerobes in the gut and soil displayed similar physiological functionalities. A total of 136 N2O-producing isolates that reduced either nitrate or nitrite were obtained from high serial dilutions of gut homogenates. Of the 25 representative N2O-producing isolates that were chosen for characterization, 22 isolates exhibited >99% 16S rRNA gene sequence similarity with their closest cultured relatives, which in most cases was a soil bacterium, most isolates were affiliated with the gamma subclass of the class Proteobacteria or with the gram-positive bacteria with low DNA G+C contents, and 5 isolates were denitrifiers and reduced nitrate to N2O or N2. The initial N2O production rates of denitrifiers were 1 to 2 orders of magnitude greater than those of the nondenitrifying isolates. However, most nondenitrifying nitrate dissimilators produced nitrite and might therefore indirectly stimulate the production of N2O via nitrite-utilizing denitrifiers in the gut. The results of this study suggest that most of the N2O emitted by earthworms is due to the activation of ingested denitrifiers and other nitrate-dissimilating bacteria in the gut lumen.