Methane inhibited NH4+ utilization by Nitrosomonas europaea with a Ki of 2mM. O2 consumption was not inhibited. In the absence of NH4+, or with hydrazine as reductant, methane caused nearly a doubling in the rate of O2 uptake. The stimulation was abolished by allylthiourea, a sensitive inhibitor of the oxidation of NH4+. Analysis revealed that methanol was being formed in these experiments, with yields approaching 1 mol of methanol per mol of O2 consumed under certain conditions. When cells were incubated with NH4+ under an atmosphere of 50% methane, 50 microM-methanol was generated in 1 h. It is concluded that methane is an alternative substrate for the NH3-oxidizing enzyme (ammonia mono-oxygenase),m albeit with a much lower affinity than for methane mono-oxygenase of methanotrophs.