The role of sulfide in the relief of acetylene inhibition of nitrous oxide reduction by Flexibacter canadensis was studied. In this organism, the reversal of acetylene inhibition of nitrous oxide reduction is correlated with a 90% decrease in the dissolved sulfide concentration. The fate of this sulfide is not known, since there was no concomitant increase in acid-soluble sulfide and volatile sulfur compounds were not detectable by flame photometric gas chromatography. Of the other sulfur-containing compounds tested (sulfate, sulfite, thiosulfate, cysteine, methionine, dithionite, dithionate, and glutathione), only cysteine relieved the acetylene block of nitrous oxide reduction by F. canadensis. Under similar experimental conditions, other denitrifiers tested (Azospirillum brasilense, Pseudomonas stutzeri, and a Flavobacterium isolate) failed to reduce nitrous oxide in the presence of sulfide and an inhibitory concentration of acetylene. It is concluded that both biological and abiological factors contribute to the sulfide relief of acetylene inhibition of nitrous oxide by pure cultures of F. canadensis.