Rift Valley fever (RVF) is an anthropozoonosis caused by a Phlebovirus (Bunyaviridae family) that has re-emerged recently in East and West Africa in 1997-1998. This emphasizes the need for early and rapid detection of the virus and an efficient surveillance system. To this goal, a single tube or a nested reverse transcriptase-polymerase chain reaction (RT-PCR) method focusing on the NSs coding region of the S segment was developed and used to detect the RVF virus (RVFV) genome, resulting respectively in the synthesis of 810 and 662 bp DNA amplimers. The assay was specific for RVFV and did not amplify any other phleboviruses known to circulate in sub-Saharan Africa. When serial dilutions of RVFV were artificially mixed with human normal serum, the minimal detection limits were 50 and 0.5 plaque forming units respectively using the simple and the nested RT-PCR. The RT-PCR method was efficient for the detection of RVFV RNA in the blood from experimentally RVFV-infected mice and lamb and the nested RT-PCR was found more sensitive than the virus isolation method. Additionally, this detection method was applied successfully for the diagnosis of human cases during the 1998 Mauritanian outbreak.