The involvement of insects in Reticuloendotheliosis virus (REV) transmission was examined by testing insects trapped at commercial farms and by controlled feeding experiments using mosquitoes, Culex pipiens L. and house flies, Musca domestica L. We established sensitive methods of REV detection, including reverse transcription-polymerase chain reaction (PCR) for REV-LTR and REV-gag genes, REV antigenemia measurements by enzyme-linked immunosorbent assay, and virus isolation in tissue cultures. A variety of blood-sucking species of insects were trapped at farms with infected poultry and tested, but none were positive. To rule out the possibility of PCR inhibition by insect RNA, spiking experiments were conducted and no interference was observed. Because Cx. pipiens mosquitoes were trapped frequently at farms, we performed feeding experiments with mosquito females fed on a REV-containing tissue culture medium and chicken blood mixture. Virus was detected in the mosquitoes up to 5 h postfeeding, compared with 96 h in the feeding mixture, indicating that Cx. pipiens can only harbor REV for a short period. House flies were suspected to be involved in the virus transmission because they frequently were trapped on positive farms. In contrast to mosquitoes, REV was harbored within the house fly digestive tract for up to 72 h and could infect chickens, as demonstrated by seroconversion and by detection of viral gag-sequence in the cloaca. The current study is supportive for the role of house flies as a mechanical vector of REV among poultry.