Many arthropod-borne viruses (arboviruses) are important human pathogens medically. The development of an effective technique to detect the viruses by using nucleic acid amplification, such as polymerase chain reaction (PCR), improves not only clinical diagnosis but also virologic surveillance of mosquito vectors in the field. In this study, the development of an improved and simplified assay is described for detection of mosquitoes infected with eastern equine encephalitis (EEE) virus, Cache Valley (CV), and California (CAL) serogroup viruses from field-collected mosquito pools. As little as 5 microl of homogenate from mosquito pools was used in the reverse transcription (RT) reaction followed by the use of three sets of specific primers for the PCR. Positive pools were determined by finding PCR bands of the expected size for each arbovirus. The confirmation and identification of Bunyaviruses was done by sequencing the PCR product. In 1999, West Nile virus (WNV) was identified as the etiologic agent of an outbreak of human encephalitis in New York City. It is shown that this protocol is also able to detect West Nile viral RNA in a pool of 100 mosquitoes containing one infected mosquito.