Autotrophic ammonia-oxidizing bacteria produce nitrous oxide (N(2)O) as a by-product of nitrification or as an intermediate of nitrifier denitrification. In soil incubations, acetylene (C(2)H(2)) and large partial pressures of oxygen (O(2)) are used to distinguish between these sources. C(2)H(2) inhibits ammonia oxidation and should therefore inhibit N(2)O production by both nitrification and nitrifier denitrification. O(2) suppresses the reduction pathway of nitrifier denitrification. However, doubts concerning the reliability of C(2)H(2) and O(2) as inhibitors have arisen recently. Therefore, in this study we tested the influence of C(2)H(2) and large partial pressures of O(2) alone and in combination on N(2)O production in pure cultures of the ammonia oxidizers Nitrosomonas europaea and Nitrosospira briensis. C(2)H(2) largely inhibited nitrite production in both ammonia oxidizers and N(2)O production by N. europaea. Surprisingly, it did not affect the N(2)O production in N. briensis. The variable response of ammonia oxidizers to C(2)H(2) might have consequences for the use of C(2)H(2) as an inhibitor of nitrification in soils. Different partial pressures of O(2) ranging from less than 10 kPa O(2) to 100 kPa O(2) were tested for their effectiveness in inhibiting N(2)O production via nitrifier denitrification. The partial pressure of 100 kPa O(2) yielded minimal N(2)O production by both ammonia-oxidizing species and seemed to inhibit N(2)O emission from nitrifier denitrification to a large extent. However, a negative effect of 100 kPa O(2) on ammonia oxidation itself could not be excluded. The applicability of both inhibitors in determining N(2)O production pathways in soils is discussed.