Objective: To study the sensitivity and specificity of 14-3-3 testing in a prospective series of patients suspected of having Creutzfeldt-Jakob disease (CJD). Background: The 14-3-3 protein immunoassay on CSF has favorable test characteristics as a premortem diagnostic tool in CJD. However, the 14-3-3 protein is a normal cellular protein expressed in various tissues, and its presence in CSF reflects extensive destruction of brain tissue as in CJD, but also in ischemic stroke and meningoencephalitis. Methods: 14-3-3 was tested in the CSF of a prospective series of 110 consecutive patients suspected of having CJD. Results: The sensitivity was 97% and the specificity was 87% in this series. False-positive results were mainly caused by stroke and meningoencephalitis. Conclusion: The 14-3-3 protein is a highly sensitive and specific marker for CJD when used in the appropriate clinical context.