OBJECTIVE To determine the test performance characteristics of serial creatine kinase-MB (CK-MB) mass measurements for acute myocardial infarction (MI) in patients presenting to the ED with chest pain and nondiagnostic ECGs. METHODS A prospective, observational test performance study was conducted. Hemodynamically stable patients aged > or = 25 years with chest discomfort, but without ECGs diagnostic for MI, were enrolled at 7 university teaching hospitals. Presenting ECGs showing > 1-mV ST-segment elevation in > or = 2 electrically contiguous leads were considered diagnostic for MI; patients with diagnostic ECGs on presentation were excluded. Real-time, serial CK-MB mass levels were obtained using a rapid serum immunochemical assay at the time of ED presentation (0-hour) and 3 hours later (3-hour). The following testing schemes were evaluated for their sensitivity and specificity for detection of MI during patient evaluation in the ED: 1) an elevated (> or = 8 ng/mL) presenting CK-MB level; 2) an elevated presenting and/or 3-hour CK-MB level; 3) a significant increase (i.e., > or = 3 ng/mL) within the range of normal limits for CK-MB concentrations during the 3-hour period (delta CK-MB); and/or 4) development of ST-segment elevation during the 3 hours (second ECG). RESULTS Of the 1,042 patients enrolled, 777 (74.6%) were hospitalized, including all 67 MI patients (8.6% of admissions). As a function of duration of time in the ED, the test performance characteristics of serial CK-MBs for MI (and cumulative data for the additional ECG) were: [table: see text] The 0-hour to 3-hour CK-MB positive and negative predictive values were 52% to 55% and 96% to 99%, respectively. The sensitivities of serial CK-MB results as a function of the interval following chest discomfort onset were: [table: see text] CONCLUSIONS Serial CK-MB monoclonal antibody mass measurements in the ED can identify MI patients with initially nondiagnostic ECGs. CK-MB sensitivity significantly increases over 3 hours of observation of stable chest discomfort patients in the ED; it also increases as a function of the total interval from onset until enzyme measurement.