Background: European data support the use of low high-sensitivity troponin (hs-cTn) measurements or a 0/1-hour (0/1-h) algorithm for myocardial infarction (MI) or to exclude major adverse cardiac events (MACE) among Emergency Department (ED) patients with possible acute coronary syndrome (ACS). However, modest US data exist to validate these strategies. This study evaluated the diagnostic performance of an initial hs-cTnT measure below the limit of quantification (LOQ: 6 ng/L), a 0/1-h algorithm, and their combination with HEART scores for excluding MACE in a multisite US cohort. Methods: A prospective cohort study was conducted at 8 US sites, enrolling adult ED patients with symptoms suggestive of ACS and without ST-elevation on electrocardiogram. Baseline and 1-hour blood samples were collected and hs-cTnT (Roche, Basel Switzerland) measured. Treating providers blinded to hs-cTnT results prospectively calculated HEART scores. MACE (cardiac death, MI, and coronary revascularization) at 30-days was adjudicated. The proportion of patients with initial hs-cTnT measures <LOQ and risk based on a 0/1-h algorithm was determined. The negative predictive value (NPV) was calculated for both strategies when used alone or with a HEART score. Results: Among 1,462 participants with initial hs-cTnT measures, 46.4% (678/1,462) were women and 37.1% (542/1,462) were African American with a mean age of 57.6 (SD±12.9) years. MACE at 30-days occurred in 14.4% (210/1,462). Initial hs-cTnT measures <LOQ occurred in 32.8% (479/1,462), yielding a NPV of 98.3% (95%CI: 96.7-99.3%) for 30-day MACE. A low risk HEART score with an initial hs-cTnT < LOQ occurred in 20.1% (294/1,462) yielding a NPV of 99.0% (95%CI: 97.0-99.8%) for 30-day MACE. A 0/1-h algorithm was complete in 1,430 patients, ruling-out 57.8% (826/1,430) with a NPV of 97.2% (95%CI: 95.9-98.2%) for 30-day MACE. Adding a low HEART score to the 0/1-h algorithm ruled-out 30.8% (441/1430) with a NPV of 98.4% (95%CI: 96.8-99.4%) for 30-day MACE. Conclusions: In a prospective multisite US cohort, an initial hs-cTnT 99% for 30-day MACE when used alone or with a HEART score. Clinical Trial Registration: URL: https://clinicaltrials.gov Unique Identifier: NCT02984436