BACKGROUND: Acute ischemic stroke remains largely a clinical diagnosis. OBJECTIVE: To assess the potential of several biomarkers to distinguish acute ischemic stroke from mimics in the emergency department (ED). METHODS: In this prospective study, 63 patients with suspected acute stroke were enrolled. Blood samples were collected at ED presentation and assayed for B-type natriuretic peptide, C-reactive protein (CRP), matrix metalloproteinase 9 (MMP-9), D-dimer, and protein S100B. Final diagnosis of stroke was rendered by blinded independent stroke experts after review of all clinical, imaging, and conventional laboratory data during admission. Logistic regression and bootstrapping models were used to evaluate the association between biomarker values and acute stroke. RESULTS: Thirty-four patients had a final diagnosis of stroke and 29 with mimics. The initial ED values of CRP, MMP-9, and S100B (C-indices of 0.808, 0.811, and 0.719, respectively) and the National Institutes of Health Stroke Scale (NIHSS) (C-index 0.887) predicted acute cerebral ischemia. CRP levels added discriminative value over clinical variables alone in the diagnosis of stroke. When the levels of CRP were added to the NIHSS, the combination was highly predictive of stroke (bootstrap mean C-index 0.951, 90% Confidence Interval 0.903-0.991, likelihood test p = 0.004). CONCLUSIONS: Biomarker testing with CRP and potentially MMP-9 and S100B, may add valuable and time-sensitive diagnostic information in the early evaluation of patients with suspected stroke in the ED. Future prospective evaluations are necessary to validate the diagnostic capability of these biomarkers for acute ischemic stroke in the ED before they should be considered for use in clinical practice.