Three different extracts of Thermoactinomyces sacchari were analyzed for their antigenicity and physical-chemical properties. Rabbit antiserum to each preparation, tested by immunodiffusion in gel, demonstrated that the most potent immunogen was that prepared by the double-dialysis method. This extract also contained the greatest number of precipitating antigens as detected by gel filtration. All extracts, analyzed by column chromatography on Sephadex G-75, were heterogeneous in that they contained large- and small-molecular-weight fractions. Precipitins in each extract were detected in column eluates of relatively low ultraviolet absorption and were of a similar molecular size range. Chemical analysis of purified antigens demonstrated protein and carbohydrate. The culture supernatant contained the greatest amount of carbohydrate (66%), and the soluble extract of bacterial cells contained the greatest amount of protein (68%). Several antigens were partially sensitive to the proteolytic enzymes Pronase and trypsin, but none was sensitive to lysozyme. These results demonstrate that there are multiple-antigen systems in T. sacchari and that, of the samples analyzed, the double-dialysis method of antigen preparation yields the most potent antigens.