We examined what kind and how much of the specific inhibitor is effective for the estimation availabilities of substrates for methanogens in anaerobic sludges. The chloroform (0.1%, v/v) inhibited 90% of the methanogenesis in an anaerobic sludge (LD), which was cultivated with glucose, and was more effective than 2-bromoethanesulfonic acid (BES, 0-200 mM). Hydrogen, acetate and propionate were linearly accumulated when the methanogenesis was inhibited by 0.1% of chloroform. The propionate accumulation rate in the presence of chloroform was identical with that in the presence of high partial pressure of hydrogen (H-2:CO2 = 20:80) which inhibits syntrophic H-2-producing fatty-acids-oxidizing bacteria. This shows that the methanogens utilized hydrogen and acetate converted by syntrophic hydrogen-producing propionate-oxidizing bacteria through interspecies hydrogen transfer. Using chloroform (0.1%) as the specific inhibitor, in the LD sludge 49% of methane was estimated to be derived from propionate. The H-2 + CO2 and acetate from the other pathway (NS), contributed 2 and 34% of the methanogenesis, respectively. This method was also applied to a municipal sewage digester sludge (MD). In the MD sludge, methanogenesis from propionate, H-2 + CO2 and acetate via the NS pathway were 16, 2 and 78%, respectively. Major substrates for methanogenesis, therefore, could be estimated in anaerobic sludges.