Anaerobic enrichment cultures with H2S and N2O as substrates which were inoculated with a biofilm sample showed rapid growth and gas formation after 2 to 3 days at 27 degrees C. By using the deep-agar dilution technique, a pure culture was obtained. The strain was tentatively identified as Thiobacillus denitrificans. The isolate was used for batch and gradient culture studies under denitrifying conditions, oxidizing H2S with concomitant reduction of N2O to N2. In batch culture, oxidation of H2S was stepwise, with transient accumulation of elemental sulfur; the final oxidation product was SO4(2-). In gradient culture, there was no notable accumulation of elemental sulfur and microsensor measurements of H2S and N2O showed that H2S was oxidized directly to SO4(2-). In the presence of C2H2, however, oxidation of H2S stopped at the level of elemental sulfur and no SO4(2-) was produced in either batch or gradient cultures. This is a hitherto unknown inhibitory effect of C2H2. The inhibition is suggested to occur at the level of sulfite reductase, which catalyzes the oxidation of elemental sulfur to SO3(2-) in T. denitrificans. However, reduction of N2O in this strain was, surprisingly, not affected by C2H2. The isolate is the first chemolithoautotrophic organism shown to reduce N2O in the presence of C2H2. Denitrification in natural ecosystems is often quantified as N2O accumulation after C2H2 addition. However, the presence of large numbers of similar organisms with C2H2-insensitive N2O reduction could lead to underestimation of in situ rates.