Fourteen hybridoma cell lines secreting antibodies against the Semliki Forest virus nucleocapsid protein were established and employed for identification of conserved epitopes among 27 alphavirus types and subtypes. Using an antibody capture test, the antibodies were found to cross-react to variable degree with alphaviruses belonging to the Semliki Forest, western encephalitis and eastern encephalitis complexes, as well as Middelburg and Ndumu. None of the antibodies reacted with either Venezuelan equine encephalitis or Barmah Forest virus. Due to their reactivity with Fort Morgan, Y62-33, Whataroa and chikungunya, the monoclonal antibodies were divided into six reactive types. Competition assays showed that the epitopes for all the types were either identical or clustered on a single domain of the nucleocapsid protein.