In eastern chipmunks (Tamias striatus) inoculated intramuscularly with 101.5 to 105.7 PFU of West Nile virus (WNV), serum titers developed sufficient to infect Aedes triseriatus (Say), Ae. vexans (Meigen), andCulex pipiens (L.). Mean titers (95% confidence interval) of 8 chipmunks were 103.9(3.3–4.5), 106.7(6.4–7.0), and 105.8(4.1–7.5) PFU/mL on days 1–3 postinoculation (p.i.) and 105.8 PFU/mL in 1 chipmunk on day 4 p.i. Mean estimated days that WNV titers were >104.8 and >105.6 were 1.7 (1.1–2.3) and 1.4 (1.0–1.6). The longest period of viremia >104.8 PFU/mL was 3–4 days. WNV antigen was detected in the small intestine of 2 chipmunks and the kidneys of 4 chipmunks by immunohistochemistry. WNV also was detected in urine, saliva, and feces of some chipmunks. These data suggest chipmunks might play a role in enzootic WNV cycles and be an amplifying host for mosquitoes that could infect humans.