Serological assays for diagnosis of Venezuelan equine encephalitis virus (VEEV) currently require biosafety level 3 facilities and select agent certification to produce antigens, reference sera, or viral stocks. Rapid identification of VEEV infection is required to respond to human and equine outbreaks of encephalitis caused by that virus and can be useful for epidemiologic surveillance. Alphavirus (Sindbis)-based recombinant viruses that express VEEV structural proteins are attenuated in animal models, thus representing an alternative to the handling of virulent infectious virus. Virus and viral antigens from recombinant Sindbis/VEE constructs engineered to express structural proteins from multiple VEEV subtypes were evaluated as diagnostic reagents in VEEV-specific serological assays, e.g., plaque reduction neutralization test (PRNT), hemagglutination inhibition (HI) assay, and complement fixation (CF) test. Chimeric viruses were produced efficiently in cell culture and were as effective as the parental virus for identifying infection of humans, horses, and rodents in these serological assays. Copyright © 2007 by The American Society of Tropical Medicine and Hygiene.