Background: Recent guidelines for use of cardiac troponin to detect cardiac damage and for cardiovascular risk stratification have made increasingly sensitive troponin assays important. Troponin assays continue to be plagued by interferences caused by heterophilic antibodies (HAs). We evaluated the performance of a revised cardiac troponin I (cTnI) assay designed to have increased analytical sensitivity and to minimize the effect of HAs. Methods: The revised Dade Behring Dimension® cTnI assay was evaluated according to NCCLS EP5-A at five institutions. Plasma samples from 14 309 patients were assayed by the original Dimension cTnI assay. To identify samples that may have interfering HAs, samples with values >1.4 μg/L were reanalyzed on the Dade Behring Stratus® CS cTnI assay. Samples with possible interfering antibodies were also analyzed before and after selective absorbance studies on the revised Dade Behring Dimension cTnI assay. Results: The limit of quantification in the revised method was 0.1 μg/L with imprecision (CV) of 11-17% at 0.1 μg/L. Values correlated well with the Stratus CS cTnI method: revised = 1.06(original) + 0.01; r = 0.98, Sy/x = 0.25 μg/L). Falsely increased results consistent with myocardial infarction by the original Dimension cTnI assay and presumably attributable to HAs were identified in 0.17% of all patients with samples submitted for cTnI analysis. The revised Dimension cTnI assay eliminated the interference in 17 of 25 samples identified and greatly decreased the interference in the other 8. Conclusions: The revised Dimension cTnI method greatly minimizes the effect of interfering HAs. It also exhibits analytical performance characteristics consistent with recent guidelines for use of this assay to detect cardiac damage.