A radioimmunoassay (RIA) procedure is described for measuring antibodies to alphaviruses in human and other mammalian sera. The test employed protein Abearing Staphylococcus aureus as a solid-phase immunoadsorbent for (3)H-labeled viruses complexed with immunoglobulin G. Using antibodies produced in humans and guinea pigs, the RIA procedure clearly differentiated among antibodies to Venezuelan, western, and eastern equine encephalomyelitis viruses. Sensitivity of the RIA depended on the concentrations of labeled viruses employed. The dilution of serum that effected binding of 50% of the (3)H-labeled virus (determined by probit analysis) was consistently higher than the neutralizing antibody titer determined by a conventional plaque reduction neutralization test using 80% plaque reduction end points. In addition, sera from 73 individuals were screened for seroconversion following live attenuated Venezuelan equine encephalomyelitis virus vaccine (strain TC-83) inoculation, by RIA using a single serum dilution (1:80); results were identical with seroconversions identified by plaque reduction neutralization test. Hyperimmune Venezuelan equine encephalomyelitis virus sera from a number of mammalian species were successfully titrated by RIA; the species tested were human, guinea pig, white rat, rabbit, burro, dog, monkey, sheep, and cotton rat. The protein A-mediated RIA is a rapid, sensitive, specific, and precise serological tool for measuring antibodies to surface antigens of alphaviruses, and should allow the subsequent development of a competitive binding RIA to measure antigenic potency of inactivated alphavirus vaccines.