Toxin preparations were obtained by growing Clostridium difficile VPI strain 10463 in 2-liter brain heart infusion dialysis flasks at 37 degrees C for 3 days. The initial step of the purification scheme involved ultrafiltration through an XM-100 membrane filter. Two toxic activities, designated toxins A and B, were separated by ion-exchange chromatography on DEAE-NaCl gradients. Toxin A was purified to homogeneity by an acetic acid precipitation at pH 5.5. Other separation techniques, including CM Sepharose CL-6B, (NH4)2SO4 and acetic acid precipitations, and hydrophobic interaction chromatography, were examined in attempts to further purify toxin B. Although these methods failed to increase the specific activity of toxin B, they provided additional evidence that the two toxins are distinct molecules. The toxins are acid and heat labile and are inactivated by trypsin and chymotrypsin, but not by amylase. The molecular weight of toxin A, as estimated by gel filtration and gradient polyacrylamide electrophoresis, ranged from 440,000 to 500,000. The estimated molecular weight of toxin B was 360,000 to 470,000.