A reverse transcription (RT) multiplex polymerase chain reaction (PCR) assay was developed to allow rapid, sensitive and simultaneous detection of enteroviral RNA and herpesviral DNA specific sequences in a single tube. The method involves a reverse transcription step followed by a multiplex nested PCR in which the combination of primers amplifies cDNA from enteroviruses and specific herpesviruses DNA. Nested amplification utilises primers designed to anneal into the amplification product from the first reaction. Individual viruses were then detected and differentiated by the size of their PCR products determined using ethidium bromide stained agarose gels. To exclude false negatives due to sample inhibitors an internal amplification control, a cloned fragment of DNA from Pseudorabies virus (PRV DNA) was included in the reaction mixture. Detection levels between 0.01 and 0.001 TCID50 of prototype strains of Polio and Coxsackie type B viruses and between 1 and 100 molecules of cloned-DNA of herpesviruses prototype strains were achieved. The RT multiplex PCR method proved capable of detecting enteroviral RNA or herpesviral DNA in cerebro spinal fluid (CSF) samples from patients with aetiologically well characterized encephalitis or aseptic meningitis.