Enterobacteriaceae share a common antigen (CA). This antigen exists as a powerful immunogen, when produced by Escherichia coli O14, and as a minimally effective immunogen, when present in cultures of most other smooth strains. The present study was directed toward the assessment of immunogenicity of this antigen produced by various rough mutants and their parent strains as well as that of viable and nonviable bacterial suspensions. Rabbits were immunized intravenously, and the passive hemagglutination test was used for the quantitation of CA antibodies. The following results were obtained. (i) Immunization of rabbits with viable or heat-killed (1 hr, 100 C) suspensions of smooth parent strains resulted in only slight or insignificant antibody production. Of seven additional smooth strains of Enterobacteriaceae tested, none induced CA antibodies upon immunization with killed (1 hr, 100 C) suspensions, and only two engendered antibodies in moderate titers when viable suspensions were used, possibly due to the presence of undetected R mutant cells. (ii) In contrast, administration of viable suspensions of the corresponding mutant strains, regardless of core types, including E. coli R1, R2, R3, and Salmonella Ra types, caused a significant antibody response. (iii) The immunogenicity of R mutants differed strikingly, inasmuch as mutants with the R1 core [E. coli O8−:K27−(R) and Shigella boydii type 3−(R)] engendered CA antibodies when heated suspensions were administered, in contrast to heated suspensions of mutants with the R2, R3, and Ra cores. (iv) Viability per se is not an abolute requirement for immunogenicity of R2 mutants, since CA antibodies were engendered by suspensions killed at 60 C or by merthiolate or formaldehyde. (v) Ethanol fractionation revealed that immunogenic CA of the R1 mutant is ethanol-insoluble and that of the parental smooth strain is ethanol-soluble; thus, CA of R1 mutants resembles that of E. coli O14 in both immunogenicity and ethanol insolubility.