Pulse-chase experiments after synchronous initiation of translation indicate that the larger Venzuelan equine encephalomyelitis (VEE) virus membrane glycoprotein E2, is derived by proteolytic cleavage of the precursor, PE2. The structural proteins of VEE virus strains representing each of the antigenic subtypes and varieties have been compared by discontinuous SDS-polyacrylamide gel electrophoresis. Nucleocapsid proteins of all isolates were similar in size (mol. wt. 35 to 36 X 10(3). The mol. wt. of E1 varied from 48 to 51 X 10(3) and the mol. wt. of E2 glycoproteins ranged from 53 to 59 X 10(3). Pixuna virus contained a third envelope glycoprotein of 59 X 10(3) mol. wt. in addition to the two major glycoproteins of mol. wt. 53 X 10(3) and 48 X 10(3) respectively. The isoelectric points (pI) of E1 and E2 for all VEE strains studied were approx. 7 and 9 respectively. Both glycoproteins of TC-83 virus induced precipitating antibodies which reacted only with the homologous purified E1 and E2 glycoproteins. Antibodies to E2 protein of each virus neutralized virus infectivity and inhibited the agglutination of goose erythrocytes by virions. Haemagglutination-inhibition tests using antisera to E2 glycoproteins of prototype viruses, representing each of the antigenic subtypes and varieties, differentiated the viruses into subtypes I, II, III and IV with subtype I divided into variants 1AB, 1C, 1D and 1E.