To better define the possible role of argasid ticks in the epidemiology of West Nile virus, adult Argas arboreus, A. persicus, and A. hermanni were fed through a membrane on fetal bovine serum containing 105.5 50% tissue culture infective doses (TCID50)/ml of West Nile virus. The virus was detected for three and four days after feeding in A. persicus and A. hermanni, respectively. The virus titers then decreased to undetectable levels in both species. When the infective dose was increased to 106.2, virus was detected until days 6 and 8, respectively. In A. arboreus, virus titers in whole tick homogenates reached a peak of 104.0 on day 4 postfeeding and remained constant at 103.0 after day 6 throughout the 20- or 50-day observation periods. Virus was detected by isolation, indirect fluorescent antibody, and histochemical techniques in the salivary glands, ovaries, synganglia, and coxal fluids. Infected ticks successfully transmitted virus to clean chickens on day 20 postfeeding. No evidence of transstadial transmission from nymph to adult was detected. Larvae from experimentally infected females successfully transmitted virus to clean chicks and virus was recovered from F1 larvae. Venereal transmission was not detected. Virus was present in coxal fluids secreted by infected females after infective meals. This study demonstrates West Nile virus infection in experimentally infected A. arboreus ticks and documents horizontal and vertical transmission in this species.