ABSTRACT The effects of three metabolic inhibitors (acetylene, methanol, and allylthiourea [ATU]) on the pathways of N ₂ production were investigated by using short anoxic incubations of marine sediment with a ¹⁵ N isotope technique. Acetylene inhibited ammonium oxidation through the anammox pathway as the oxidation rate decreased exponentially with increasing acetylene concentration; the rate decay constant was 0.10 ± 0.02 μM ⁻¹ , and there was 95% inhibition at ∼30 μM. Nitrous oxide reduction, the final step of denitrification, was not sensitive to acetylene concentrations below 10 μM. However, nitrous oxide reduction was inhibited by higher concentrations, and the sensitivity was approximately one-half the sensitivity of anammox (decay constant, 0.049 ± 0.004 μM ⁻¹ ; 95% inhibition at ∼70 μM). Methanol specifically inhibited anammox with a decay constant of 0.79 ± 0.12 mM ⁻¹ , and thus 3 to 4 mM methanol was required for nearly complete inhibition. This level of methanol stimulated denitrification by ∼50%. ATU did not have marked effects on the rates of anammox and denitrification. The profile of inhibitor effects on anammox agreed with the results of studies of the process in wastewater bioreactors, which confirmed the similarity between the anammox bacteria in bioreactors and natural environments. Acetylene and methanol can be used to separate anammox and denitrification, but the effects of these compounds on nitrification limits their use in studies of these processes in systems where nitrification is an important source of nitrate. The observed differential effects of acetylene and methanol on anammox and denitrification support our current understanding of the two main pathways of N ₂ production in marine sediments and the use of ¹⁵ N isotope methods for their quantification.