OBJECTIVE: To determine humoral responses to an equine encephalitis vaccine in healthy alpacas. DESIGN: Clinical trial. ANIMALS: 39 healthy alpacas on 1 farm and 86 healthy alpacas on a second farm. PROCEDURES: All alpacas were given 3 doses IM of a bivalent, killed-virus equine encephalitis vaccine, with 4 weeks between doses. Eastern equine encephalitis (EEE) virus neutralizing antibody responses were determined with a plaque reduction neutralization assay every 14 days in alpacas on the first farm and 70 days after the first dose of vaccine on the second farm. RESULTS: For alpacas on the first farm, geometric mean virus neutralizing antibody titer peaked 2 weeks after the third vaccine dose was given (ie, day 70). At this time, 29 of 38 (76%) animals were seropositive for antibodies against EEE virus, and percentage of animals 6 years old that were seropositive (1/5). For alpacas on the second farm, 76 (88%) were seropositive on day 70, and percentage of animals 6 years old that were seropositive (27/33). For both farms, geometric mean titer on day 70 was significantly higher in animals < 2 years old than in animals > 6 years old. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that inoculation of alpacas with 3 doses of a bivalent, killed-virus equine encephalitis vaccine induced a humoral antibody response against EEE virus.