We developed a method based on a coupled reverse transcription-PCR (RT- PCR) for the detection of Lassa virus using primers specific for regions of the S RNA segment which are well conserved between isolates from Sierra Leone, Liberia, and Nigeria. The specificity of the assay was confirmed by Southern blotting with a chemiluminescent probe. The assay was able to detect 1 to 10 copies of a plasmid or an RNA transcript containing the target sequence. There was complete concordance between RT-PCR and virus culture for the detection of Lassa virus in a set of 29 positive and 32 negative serum samples obtained on admission to the hospital from patients suspected of having Lassa fever in Sierra Leone. Specificity was confirmed by the failure of amplification of specific products from serum samples collected from 129 healthy blood donors in Sierra Leone or from tissue culture supernatants from cells infected with related arenaviruses (Mopeia, lymphocytic choriomeningitis, Tacaribe, and Pichinde viruses). Sequential serum samples from 29 hospitalized patients confirmed to have Lassa fever were tested by RT-PCR and for Lassa virus-specific antibodies by indirect immunofluorescence (IF). RT-PCR detected virus RNA in 79% of the patients at the time of admission, comparing favorably with IF, which detected antibodies in only 21% of the patients. Lassa virus RNA was detected by RT-PCR in all 29 patients by the third day of admission, whereas antibody was detectable by IF in only 52% of the patients. These results point to an important role for RT-PCR in the management of suspected cases of Lassa fever.