In febrile patients, distinguishing bacterial from viral infections is crucial for early treatment initiation and rational use of antibiotics. Raised interferon-alpha (IFN-alpha) levels in serum has been associated with a wide range of viral infections. We evaluated the effectiveness of IFN-alpha serum measurements for the etiological diagnosis of febrile patients. Adult patients who were attending the emergency department with body temperature above or equal to 38.5 degrees C were studied prospectively, followed-up until day 30, and classified by two independent experts (blind for IFN-alpha results) as having a bacterial/parasitic infection, viral infection, or other diagnosis. The results of IFN-alpha measurements in blood samples taken in the emergency room, were compared with expert diagnosis. Among 243 patients included, 167 had bacterial/parasitic infections (including 19 with viral co-infection), 59 had viral infections, and 36 other diagnoses. IFN-alpha assay had a sensitivity of 0.44 and a specificity of 0.92 for the diagnosis of viral infection. Among the 20 patients with acute viral infection according to the emergency physician diagnosis, 7 (35%) were given antibiotics, including four patients with raised IFN-alpha concentrations. It is concluded that in febrile patients, raised serum IFN-alpha level is highly specific of the viral etiology of fever but poorly sensitive. Reliable viral and bacterial biological markers are needed in order to improve rational use of antibiotics.