Abstract Background Symptoms of acute coronary syndromes (ACS) account for a large proportion of emergency department (ED) visits and hospitalizations. High-sensitivity troponin can rapidly rule-out or rule-in acute myocardial infarction (AMI) within a short time of ED arrival. We sought to validate test characteristics and classification performance of two-hour high-sensitivity troponin T (hsTnT) algorithms for the rapid diagnosis of AMI. Methods We included consecutive patients from four academic EDs with suspected cardiac chest pain who had hsTnT assays performed two hours apart (+/- 30 minutes) as part of routine care. The primary outcome was AMI at 7 days. Secondary outcomes included major adverse cardiac events (MACE: mortality, AMI and revascularization). Test characteristics and classification performance for multiple 2-hour algorithms were quantified. Results 722 patients met inclusion criteria. 7-day AMI incidence was 10.9% and MACE incidence was 13.7%. A two-hour rule-out algorithm proposed by Reichlin and colleagues ruled out AMI in 59.4% of patients with 98.7% sensitivity and 99.8% NPV. The two-hour rule-out algorithm proposed by the UK National Institute for Health and Care Excellence ruled out AMI in 50.3% of patients with similar sensitivity and NPV. Other exploratory algorithms had similar sensitivity but marginally better classification performance. Reichlin et al’s two-hour rule-in algorithm ruled in AMI in 16.5% of patients with 92.4% specificity and 58.5% PPV. Conclusion 2-hour hsTnT algorithms can rule-out AMI with very high sensitivity and NPV. The algorithm developed by Reichlin et al. had superior classification performance. Reichlin and colleagues' 2-hour rule-in algorithm had poor PPV and may not be suitable for early rule-in decision-making.