s Abstract The anthropogenic production of greenhouse gases and their consequent effects on global climate have garnered international attention for years. A remaining challenge facing scientists is to unambiguously quantify both sources and sinks of targeted gases. Microbiological metabolism accounts for the largest source of nitrous oxide (N 2 O), mostly due to global conversion of land for agriculture and massive usage of nitrogen-based fertilizers. A most powerful method for characterizing the sources of N 2 O lies in its multi-isotope signature. This review summarizes mechanisms that lead to biological N 2 O production and how discriminate placement of 15 N into molecules of N 2 O occurs. Through direct measurements and atmospheric modeling, we can now place a constraint on the isotopic composition of biological sources of N 2 O and trace its fate in the atmosphere. This powerful interdisciplinary combination of biology and atmospheric chemistry is rapidly advancing the closure of the global N 2 O budget.