Objective: The aim of this study was to confirm previous observations that proenkephalin A (PENK-A) may serve as prognostic marker in the setting of acute ischemic stroke in a large stroke cohort. Methods: The plasma concentration of PENK-A was measured within 72 hours of symptom onset in 320 consecutively enrolled patients with stroke. The primary outcome measures were unfavorable functional outcome (modified Rankin Scale score 0-2 vs 3-6) and mortality within 90 days. Logistic and cox proportional regression analyses were fitted to estimate odds ratios (ORs), hazard ratios (HRs) and 95% confidence intervals (CIs), respectively, for the association between PENK-A and the primary outcome measures. Results: After adjusting for demographic and vascular risk factors, PENK-A was neither independently associated with functional outcome (OR: 1.29, 95% CI: 0.16-10.35) nor mortality (HR: 1.02, 95% CI: 0.14-7.33). Conclusion: Among patients with acute stroke, PENK-A does not serve as an independent prognostic marker in this external validation cohort.