Methanogenesis and homoacetogenesis occur simultaneously in the hindguts of almost all termites, but the reasons for the apparent predominance of methanogenesis over homoacetogenesis in the hindgut of the humivorous species is not known. We found that in gut homogenates of soil-feeding Cubitermes spp., methanogens outcompete homoacetogens for endogenous reductant. The rates of methanogenesis were always significantly higher than those of reductive acetogenesis, whereas the stimulation of acetogenesis by the addition of exogenous H(2) or formate was more pronounced than that of methanogenesis. In a companion paper, we reported that the anterior gut regions of Cubitermes spp. accumulated hydrogen to high partial pressures, whereas H(2) was always below the detection limit (<100 Pa) in the posterior hindgut, and that all hindgut compartments turned into efficient H(2) sinks when external H(2) was provided (D. Schmitt-Wagner and A. Brune, Appl. Environ. Microbiol. 65:4490-4496, 1999). Using a microinjection technique, we found that only the posterior gut sections P3/4a and P4b, which harbored methanogenic activities, formed labeled acetate from H(14)CO(3)(-). Enumeration of methanogenic and homoacetogenic populations in the different gut sections confirmed the coexistence of both metabolic groups in the same compartments. However, the in situ rates of acetogenesis were strongly hydrogen limited; in the P4b section, no activity was detected unless external H(2) was added. Endogenous rates of reductive acetogenesis in isolated guts were about 10-fold lower than the in vivo rates of methanogenesis, but were almost equal when exogenous H(2) was supplied. We conclude that the homoacetogenic populations in the posterior hindgut are supported by either substrates other than H(2) or by a cross-epithelial H(2) transfer from the anterior gut regions, which may create microniches favorable for H(2)-dependent acetogenesis.