The link between similarity in amino acid sequence,for ammonia monooxygenase (AMO) and isotopic discrimination for ammonia oxidation (εAMO) was investigated in β-subdivision ammonia-oxidizing bacteria. The isotope effects for ammonia oxidation in pure cultures of the nitrifying strains. Nitrosomonas marina, Nitrosomonas C-113a, Nitrosospira tenuis, Nitrosomonas europaea, and Nitrosomonas eutropha ranged from 14.2‰ to 38.2‰. The differences in isotope effects could not be readily explained by differential rates of ammonia oxidation, transport of NH4+, or accumulation of NH2OII or N2O among the strains. The major similarities and differences observed in εAMO are, however, paralleled by similarities and differences in amino acid sequences for the α-subunit of AMO (AmoA). Robust differences in εAMO among nitrifying bacteria may be expected to influence the stable isotopic signatures of nitrous oxide (N2O) produced in various environments.