Archaeal genes for ammonia oxidation are widespread in the marine environment, but direct physiological evidence for ammonia oxidation by marine archaea is limited. We report the enrichment and characterization of three strains of pelagic ammonia-oxidizing archaea (AOA) from the North Pacific Ocean that have been maintained in laboratory culture for over 3 years. Phylogenetic analyses indicate the three strains belong to a previously identified clade of water column-associated AOA and possess 16S ribosomal RNA genes and ammonia monooxygenase subunit a (amoA) genes highly similar (98-99% identity) to those recovered in DNA and complementary DNA clone libraries from the open ocean. The strains grow in natural seawater-based liquid medium while stoichiometrically converting ammonia (NH(3)) to nitrite (NO(2)(-)). Ammonia oxidation by the enrichments is only partially inhibited by allylthiourea at concentrations known to completely inhibit cultivated ammonia-oxidizing bacteria. The three strains were used to determine the nitrogen stable isotope effect ((15)ɛ(NH3)) during archaeal ammonia oxidation, an important parameter for interpreting stable isotope ratios in the environment. Archaeal (15)ɛ(NH3) ranged from 13‰ to 41‰, within the range of that previously reported for ammonia-oxidizing bacteria. Despite low amino acid identity between the archaeal and bacterial Amo proteins, their functional diversity as captured by (15)ɛ(NH3) is similar.