Nitrous oxide (N2O) plays important roles in atmospheric chemistry both as a greenhouse gas and in stratospheric ozone depletion. Isotopic measurements of N2O have provided an invaluable insight into understanding its atmospheric sources and sinks. The preference for 15N fractionation between the central and terminal positions (the “site preference”) is particularly valuable because it depends principally on the processes involved in N2O production or consumption, rather than the 15N content of the substrate from which it is formed. Despite the value of measurements of the site preference, there is no internationally recognized standard reference material of accurately known and accepted site preference, and there has been some lack of agreement in published studies aimed at providing such a standard. Previous work has been based on isotope ratio mass spectrometry (IRMS); in this work we provide an absolute calibration for the intramolecular site preference of 15N fractionation of working standard gases used in our laboratory by a completely independent technique—high-resolution Fourier transform infrared (FT-IR) spectroscopy. By reference to this absolute calibration, we determine the site preference for 25 samples of tropospheric N2O collected under clean air conditions to be 19.8‰ ± 2.1‰. This result is in agreement with that based on the earlier absolute calibration of Toyoda and Yoshida (Toyoda, S. and Yoshida, N. Anal. Chem. 1999, 71, 4711−4718) who found an average tropospheric site preference of 18.7‰ ± 2.2‰. We now recommend an interlaboratory exchange of working standard N2O gases as the next step to providing an international reference standard.