BACKGROUND: Current guidelines for the diagnosis and risk assessment of patients presenting with myocardial infarction recommend a single decision cut-off point for cardiac troponin (cTn) based on the 99th centile of a reference population. The 99th centile level for eight troponin assays was determined in an apparently cardio-healthy Australian reference population. METHODS: Nine laboratories measured troponin in serum and plasma collected from 111 reference individuals. An imprecision profile was determined using up to 10 serum samples analysed on 10 separate days. Method comparison using 100 routinely tested plasma samples was performed to estimate method concordance. RESULTS: Generally 99th centile values determined in this study were lower than, or the same as manufacturers' levels, except for cTnI by Architect (0.020 vs. 0.012 microg/L), and imprecision at the 99th centile was 20% coefficient of variation (CV) or higher. Troponin concentrations at 10% CV were greater than those quoted in the manufacturer's package insert except by AxSYM, 0.06 vs. 0.16 microg/L cTnI, and by E-170, 0.02 vs. 0.03 microg/L cTnT. In the method comparison 74, 70, 65, 75, 58, 66, 58 and 77 samples measured by Access, Architect, AxSYM, Centaur, Dimension RxL, E-170, i-STAT and Vitros ECi assays, respectively, had troponin concentrations above the study 99th centile. CONCLUSIONS: Depending on the selected reference population for troponin, there is likely to be variability in the 99th centile as shown in this study. Some differences in sample concordance at the 99th centile cut-off were observed between cTn methods and may result in different clinical classification.