Optimal conditions for preparation and storage of haemolysin were determined using spirochaetes harvested from rabbit serum broth. Haemolysin was purified by fractionation on Whatman DEAE Cellulose and Sephadex G100, purity being assessed by SDS-PAGE. The molecular weight of the haemolysin was estimated by gel filtration to be 19,000 daltons although analysis on SDS-PAGE suggested that the molecular weight of haemolysin dissociated from the RNA-core carrier was much lower. The purified haemolysin was not antigenic. The unpurified haemolysin caused lysis of several species of erythrocyte and was cytotoxic towards a range of cell monolayers, embryo bovine lung fibroblasts being the most sensitive. The cytotoxic activity of the haemolysin was quantified using a 51Chromium release assay. Of the porcine inflammatory cells tested, lymphocytes were the most sensitive. After purification the preparation was haemolytic and leucotoxic but was less cytotoxic. Lower concentrations of haemolysin were produced by an avirulent strain of T. hyody4enteniae than from the virulent strain and it was not toxic for embryo bovine lung fibroblasts. No toxic effect of haemolysin from virulent strains of T. hyody4entetiae was demonstrated either by inoculation of high concentrations into ligated colonic loops in pigs or by intragastric inoculation of CFI mice. The treponemal haemolysin is similar to Streptolysin S in the requirement for a carrier molecule to demonstrate invino haemol. ysis, and with respect to molecular weight, lack of antigenicity, cytotoxic activity and the effect on lymphocytes. When T. hyodyzente4iaew as incubated with excised colonic tissue slices and inoculated into ligated colonic loops in pigs, spirochaetes associated with the mucus rather than attaching to the colonic epithelium. Within 2 hours, spirochaetes were observed in the bottom of crypts suggested that a chemotactic mechanism may be more important than attachment in enabling T. hyodyzenten£ae to establish in the colon. iii