Abstract: Fermenter Culture supernatants of Corynebacterium pseudotuberculosis strain 3450 NCTC grown in a continuous culture were tested for cytotoxicity in 7 cell lines. The supernatants were toxic to EBL, MDCK, NBL, CHO-K1, 3T3, BHK and Vero cells. The cytotoxic activity was used as an assay method to determine the optimal incubation temperature and pH for cytotoxin production in fermenter. Fermenter culture supernatants of a reference strain C. pseudotuberculosis and three field isolates were compared for their cytotoxicity to BHK cells. Two of the field isolates were non-cytotoxic to BHK cells, one isolate had weak cytotoxicity and the reference strain was the most cytotoxic. SDS-PAGE of fermenter culture supernatants of the 4 strains revealed 35 to 39 bands with molecular weights ranging from 24 to 149 KDa.