A combination of molecular and activity measures was used to explore seasonal patterns of distribution, activity, and diversity of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in relationship to ammonia availability in Hood Canal, a fjord within the Puget Sound, Washington State estuary system. A greater contribution of AOA to nitrification, relative to AOB, was supported by complementary quantification of transcripts of a gene (amoA) coding for one subunit of the ammonia monooxygenase, quantification of intact glycerol dialkyl glycerol tetraether lipids, and kinetic modeling. High AOA: AOB ratios and nitrification rates measured by 15NH+4 dilution technique were associated with low concentrations of ammonia, whereas transient increases in AOB were most closely associated with regionally and seasonally elevated concentrations of ammonia in the water column. Additionally, single cell nitrification rates and transcript numbers were calculated for AOA and AOB populations. Together these observations offer additional ecological support for the idea that niche separation of AOA and AOB is in part determined by ammonia concentrations in estuarine ecosystems. © 2014, by the Association for the Sciences of Limnology and Oceanography, Inc.