The Lactobacillus group E antigen is a cell wall glycerol teichoic acid. The cellular locations of the group E antigen were determined in a total of 37 L. brevis strains, of which 34 strains possess S-layer proteins and three strains are S-layer protein-deficient. The group E antibody was readily and reproducibly produced only by S-layer protein-deficient Lactobacillus brevis JCM 1170 (ATCC 367). All 37 strains tested were shown to possess the group E antigen by precipitation reactions in which,whole cell extracts and group E antiserum were used. Although the three S-layer protein-deficient strains had an agglutination titer of 2048 or 4096, the 34 S-layer protein-carrying strains had low agglutination titers, ranging from 1 to 128, as determined in agglutination reactions in which intact cells and the group E antibody were used. Treatment of L. brevis strains with sodium hydroxide solutions of between 0.005 and 0.1 M resulted in removal of the S-layer protein. After removal of the S-layer proteins of the 34 S-layer protein-carrying strains by treatment of the cells with a 0.1 ha sodium hydroxide solution, the S-layer protein-stripped cells showed an agglutination titer of 2048 or 4096. These results indicate that the group E antigen is exposed on the cell surface in S-layer protein-deficient strains, but that it is masked by the S-layer proteins in strains which possess them. The group E antigen of Lactobacillus buchneri strains classified as serological group E was also shown to be located beneath S-layer proteins. The group E antibody showed marked cross reactivity with a group D lactobacillus, L. plantarum, in precipitation reactions. The extent of this cross reactivity was effectively reduced in an immunoassay with a dilute alkali-treated cells and dilute group E antiserum.