The distribution of characterized antigens in extracts of representative strains of Brucella abortus biotypes 1 to 9 was investigated by hemagglutination and precipitation reactivity. An antigen designated X was present in high concentrations in sodium dodecyl sulfate extracts of all smooth strains examined, but in low concentrations in the rough strain 45/20 extract. Antigen beta was demonstrable in 8 out of 12 smooth strains of B. abortus, but antigen gamma was detected in only 3 of these strains. Vaccine strain 19 extract contained beta antigen but no detectable gamma antigen, whereas both these antigens were present in rough strain 45/20 at slightly lower concentrations that in strain 544/W extract. Immunoenzyme histochemical studies performed with an electron microscope showed that antigens beta, gamma, and X were all situated in the outer membrane envelope of the cell wall of B. abortus 544/W.