Protein antigens eliciting delayed type hypersensitivity (DTH) were analyzed and purified from the supernatants of protein-free cultures in which Salmonella typhimurium TV148 organisms had grown. DTH activity was measured by the footpad swelling test in mice immunized with living organisms of S. typhimurium TV148 or Escherichia coli K-12. DTH activity in the culture supernatant was specific to TV148-immunized mice. This activity was destroyed by pronase. DTH activity was unable to pass through an ultrafilter with an exclusion limit of 10 kD. After condensation of the supernatant and following centrifugation (100,000 g for 1 h), the DTH activities of the sediment and the supernatant were examined, and both showed DTH activity. Further analyses of DTH antigens in the supernatant by HPLC gel filtration separated the activity into three portions. The most active portion was further fractionated by hydroxyapatite HPLC, revealing the presence of two DTH antigens, with molecular weights of 65 and less than 10 kD. These results indicate that the culture supernatant of S. typhimurium TV148 organisms contains a variety of macromolecular protein DTH-eliciting antigens, and one of the antigens is 65 kD, which is dissociated partly by organic solvents into a low molecular weight (less than 10 kD) antigen.