Acetylene (C₂H₂) inhibition technique is the most commonly applied method in potential denitrification assay. One lake sediment and three soils for rice production were used in this study to test the effectiveness of inhibition of N₂O reduction to N₂ by C₂H₂ using ¹⁵N tracer. The three rice soils were pre-incubated under anaerobic conditions to allow time for soil denitrifying enzymes, especially N₂O reduction enzyme, to be fully developed. Results show that in presence of C₂H₂, significant isotopic enrichment of ¹⁵N₂ was found at end of typical denitrification incubation. The d¹⁵N₂ value increased from the initial 0‰ to the final 7.8 -19.3‰ and 7.5 -10.6‰ for the treatment with 0.05 and 0.2 mg N labeled nitrate, respectively. Such ¹⁵N enrichment can be interpreted as N₂ formation on average accounting for 18% and 12% of the total added N in the above two treatments, respectively. Nitrous oxide accumulation in presence of C₂H₂ could not account for the total added N. Incomplete inhibition of N₂O reduction might be largely responsible for this observation. The results also indicated that part of the added nitrate was probably converted to ammonium by dissimilatory nitrate reduction reaction during the incubation. This study presents clear evidence of incomplete inhibition of N₂O reduction to N₂ by C₂H₂ in denitrification study.  Nitrogen gas formation in presence of C₂H₂ challenges validity of any C₂H₂ based techniques in denitrification study, because the denitrification rate will be underestimated if N₂O reduction to N₂ is not completely inhibited.