To evaluate the importance of eastern cottontails (Sylvilagus floridanus) as amplifying hosts for Cache Valley virus (CVV), we tested hunter-provided blood samples from northern Indiana for specific neutralizing (N) antibodies against this mosquito-borne bunyavirus. Samples were collected during the winter of 1994–95. Two seronegative eastern cottontails, captured in July 1995, were also infected with CVV by subcutaneous inoculation, and two others were infected by allowing CVV-infected mosquitoes to feed on them. The results indicate that eastern cottontails probably are not important amplifying hosts for CVV. The prevalence of N antibodies against CVV was low (6.0%, n=82) among the hunter-killed animals. Low viremia (<1.8 log₁₀ plaque-forming units/ml) of short duration (1–3 days) were seen in three of four experimentally infected eastern cottontails. The viremias were insufficient for infecting Coquillettidia perturbans, a mosquito species commonly found naturally infected with CVV.