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The δ¹⁸O value of nitrate produced during nitrification (δ¹⁸O_NO3,nit) was measured in experiments designed to mimic oceanic conditions, involving cocultures of ammonia‐oxidizing bacteria or ammonia‐oxidizing archaea and nitrite‐oxidizing bacteria, as well as natural marine assemblages. The estimates of ranged from −1.5‰ ± 0.1‰ to +1.3‰ ± 1.4‰ at δ¹⁸O values of water (H₂O) and dissolved oxygen (O₂) of 0‰ and 24.2‰ vs. Vienna Standard Mean Ocean Water, respectively. Additions of ¹⁸O‐enriched H₂O allowed us to evaluate the effects of oxygen (O) isotope fractionation and exchange on . Kinetic isotope effects for the incorporation of O atoms were the most important factors for setting overall values relative to the substrates (O₂ and H₂O). These isotope effects ranged from +10‰ to +22‰ for ammonia oxidation (O₂ plus H₂O incorporation) and from +1‰ to +27‰ for incorporation of H₂O during …