The definitive diagnosis of Creutzfeldt-Jakob disease (CJD) requires brain tissue analysis. A positive assay for the 14-3-3 protein in CSF has been suggested to be highly sensitive and specific in patients with CJD. The authors describe three patients for whom CSF 14-3-3 assays were falsely positive or falsely negative. Caution against overreliance on this putative biomarker is suggested in the diagnosis of CJD.