AbstractRNA sequences of five flaviviruses were detected by a modified polymerase chain reaction (PCR) that incorporated a reverse transcriptase and RNase inhibitor. Oligonucleotide primer pairs were synthesized to amplify sequences from St. Louis encephalitis (SLE), Japanese encephalitis (JBE), yellow fever (YF), dengue 2 (DEN‐2), and dengue 4 (DEN‐4) viruses. The amplified products were visualized as bands of appropriate size on ethidium bromide‐stained agarose gels. The identity of these products was confirmed by restriction endonuclease cleavage to generate fragments of predicted lengths. The reverse transcriptase PCR (RT‐PCR) successfully amplified flavivirus sequences from cell cultures, frozen brain tissue, and formalin‐fixed, paraffin‐embedded brain tissue. The reactions were highly specific, and the method compared favorably to two conventional assays of viral infectivity. RT‐PCR followed by PCR with nesting primers (N‐PCR) was 1,000‐fold more sensitive in detecting virus than classical infectivity titration by intracerebral inoculation of suckling mice and nearly 1,000‐fold more sensitive than amplification of virus in cell culture followed by inoculation of mice.