The nucleoprotein of Lassa virus, strain Josiah, was expressed inEscherichia coli as an N-terminally truncated, histidine-tagged recombinant protein. Following affinity purification the protein was completely denatured and spotted onto nitrocellulose membrane. A total of 1 μg of protein was applied for detection of Lassa virus antibodies (LVA) in a simple immunoblot assay. Specific anti-Lassa immunoglobulin M (IgM) antibodies could be detected by increasing the amount of protein to 5 μg. A panel of 913 serum specimens from regions in which Lassa virus was endemic and from regions in which Lassa virus was not endemic was used for evaluating the sensitivity and specificity of the LVA immunoblot in comparison to those of an indirect immunofluorescence (IIF) assay. The sera originated from field studies conducted in the Republic of Guinea (570 serum samples) and Liberia (99 serum samples), from inpatients of the clinical department of the Bernhard-Nocht-Institute, Hamburg, Germany (94 serum samples), and from healthy German blood donors (150 serum samples). In comparison to the IIF assay the LVA immunoblot assay had a specificity of 90.0 to 99.3%, depending on the origin of the specimens. The sensitivity was found to be highest for the Guinean samples (90.7%) and was lower for the Liberian samples (75%). Acute Lassa fever was diagnosed by PCR in 12 of 59 (20.3%) patients with fever of unknown origin (FUO) from the Republic of Guinea. On admission to the hospital, nine Lassa fever patients (75%) were reactive by the IgM immunoblot assay. One of the patients was infected with a new Lassa variant, which showed 10.4% variation on the amino acid level in comparison to the prototype strain of Lassa virus, Josiah. Seven PCR-negative patients were reactive by immunoblotting. The positive and negative predictive values of a single IgM immunoblot result for acute, PCR-confirmed Lassa fever were therefore 53.6 and 93.0%, respectively. Because of its high negative predictive value, a single IgM immunoblot result will be valuable for excluding acute Lassa fever for cases of FUO in areas where Lassa fever is endemic.