A serologic survey and experimental virus transmission studies were done to assess the role of domestic animals as amplifier hosts of La Crosse (LACV) and Jamestown Canyon (JCV) viruses. Serum from 319 cows, 88 dogs, 122 equines, 47 swine, 10 goats, and 4 cats were tested for neutralizing antibody to LACV, JCV, trivittatus (TVTV), and snowshoe hare (SSHV) viruses. Antibody prevalences of LACV, TVTV, and SSHV were less than 10% in all species. Antibody to JCV was detected in all species except cats. Prevalence ranged from 10% in goats and swine to 29% in dogs. No age-associated trends in JCV prevalence were noted. Two of 6 adult dogs, and 2 of 4 pigs inoculated with 6.3–6.5 log10 suckling mouse intracerebral 50% lethal doses (SMICLD50) of LACV developed viremias ranging of <1.0–2.9 log10 SMICLD50/ml 1–3 days after inoculation. Of 4 puppies inoculated with LACV, 3 developed fatal infections. Viremias were not detected in 4 cows, 4 ponies, 7 cats, or 6 sheep. Two cats fed LACV infected suckling mice shed virus from the oropharynx for 1 day each. All animals except 1 cow, 1 cat, and 1 sheep had ≥ 4-fold rise in antibody titers. Five additional dogs fed upon by LACV-infected Aedes triseriatus mosquitoes did not develop viremias or antibody and uninfected Ae. triseriatus engorging on the dogs 1–5 days after feeding by infected mosquitoes failed to become infected. Five ponies, 6 calves, 2 ewes, 6 dogs, and 5 piglets were inoculated with 3.6–7.3 log10 SMICLD50 of JCV. None developed detectable viremias, although ≥ 4-fold rises in antibody titers developed in 60% of the ponies, 17% of the calves, 50% of the dogs, and 1 of 2 ewes. None of the pigs developed corresponding rises in antibody titers. We conclude that juvenile and adult animals of the species tested are not efficient amplifier hosts of LACV or JCV, but may be useful sentinels of local virus transmission.