Natural abundance ratios of isotopocules, molecules that have the same chemical constitution and configuration, but that only differ in isotope substitution, retain a record of a compound's origin and reactions. A method to measure isotopocule ratios of nitrous oxide (N O) has been established by using mass analysis of molecular ions and fragment ions. The method has been applied widely to environmental samples from the atmosphere, ocean, fresh water, soils, and laboratory-simulation experiments. Results show that isotopocule ratios, particularly the N-site preference (difference between isotopocule ratios N N O/ N N O and N N O/ N N O), have a wide range that depends on their production and consumption processes. Observational and laboratory studies of N O related to biological processes are reviewed and discussed to elucidate complex material cycles of this trace gas, which causes global warming and stratospheric ozone depletion. © 2015 Wiley Periodicals, Inc. Mass Spec Rev 36:135-160, 2017.