IMPORTANCE Physicians need information on how to use the first available high-sensitivity troponin (hsTnT) assay in the United States to identify patients at very low risk for 30-day adverse cardiac events (ACE). OBJECTIVE To determine whether a negative hsTnT assay at 0 and 3 hours following emergency department presentation could identify patients at less than 1% risk of a 30-day ACE. DESIGN, SETTING, AND PARTICIPANTS A prospective, observational study at 15 emergency departments in the United States between 2011 and 2015 that included individuals 21 years and older, presenting to the emergency department with suspected acute coronary syndrome. Of 1690 eligible individuals, 15 (no cardiac troponin T measurement) and 320 (missing a 0-hour or 3-hour sample) were excluded from the analyses. EXPOSURES Serial hsTnT measurements (fifth-generation Roche Elecsys hsTnT assay). MAIN OUTCOMES AND MEASURES Serial blood samples from each patientwere collected after emergency department presentation (once identified as a potential patient with acute coronary syndrome) and 3 hours, 6 to 9 hours, and 12 to 24 hours later. Adverse cardiac events were defined asmyocardial infarction, urgent revascularization, or death. The upper reference level for the hsTnT assay, defined as the 99th percentile, was established as 19 ng/L in a separate healthy US cohort. Patients were considered ruled out for acutemyocardial infarction if their hsTnT level at 0 hours and 3 hours was less than the upper reference level. Gold standard diagnoses were determined by a clinical end point committee. Evaluation of assay clinical performance for acutemyocardial infarction rule-out was prespecified; the hypothesis regarding 30-day ACE was formulated after data collection. RESULTS In 1301 healthy volunteers (50.4%women; median age, 48 years), the upper reference level was 19 ng/L. In 1600 patients with suspected acute coronary syndrome (48.4%women; median age, 55 years), a single hsTnTlevel less than 6 ng/L at baseline had a negative predictive value for AMI of 99.4%. In 974 patients (77.1%) with both 0-hour and 3-hour hsTnT levels of 19 ng/L or less, the negative predictive value for 30-day ACE was 99.3%(95%CI, 99.1-99.6). Using sex-specific cutpoints, C statistics for women (0.952) and men (0.962) were similar for acutemyocardial infarction. CONCLUSIONS AND RELEVANCE A single hsTnT level less than 6 ng/L was associated with a markedly decreased risk of AMI, while serial levels at 19 ng/L or less identified patients at less than 1% risk of 30-day ACE.