Competitive oxygen kinetic isotope effects (18O KIEs) on water oxidation initiated by ruthenium oxo (RuO) complexes are examined here as a means to formulate mechanisms of O-O bond formation, which is a critical step in the production of "solar hydrogen". The kinetics of three structurally related catalysts are investigated to complement the measurement and computation of 18O KIEs, derived from the analysis of O 2 relative to natural abundance H2O under single and multi-turnover conditions. The findings reported here support and extend mechanistic proposals from 18O tracer studies conducted exclusively under non-catalytic conditions. It is shown how density functional theory calculations, when performed in tandem with experiments, can constrain mechanisms of catalytic water oxidation and help discriminate between them. © The Royal Society of Chemistry 2014.