Oxidative transformations using molecular oxygen are widespread in nature but remain a major challenge in chemical synthesis. Limited mechanistic understanding presents the main obstacle to exploiting O-2 in "bioinspired" industrial processes. Isotopic methods are presently being applied to characterize reactions of natural abundance O-2 including its coordination to reduced transition metals and cleavage of the O-O bond. This review describes the application of competitive oxygen-18 isotope effects, together with Density Functional Theory, to examine O-2 reductive activation under catalytically relevant conditions. The approach should be generally useful for probing small-molecule activation by transition-metal complexes.