Vancomycin and cholestyramine have been utilized both alone and in combination for the treatment of antibiotic-associated pseudomembranous colitis. Previous work has demonstrated significant binding of vancomycin by the anionexchange resin. The antibacterial activity of vancomycin was markedly reduced when the suspension was centrifuged and the supernatant was tested for antibacterial activity. This study confirmed these findings but demonstrated that there was no immediate loss of antibacterial activity of bound vancomycin. The degree of inactivation appeared to be dependent upon the duration of incubation of vancomycin and cholestyramine in the testing system. Antibiotic-associated pseudomembranous colitis (PMC) has been linked to the presence of Clostridium difficile within the colonic flora of patients with the disease. The organism elaborates a cytotoxin which is capable of producing the lesions found in PMC (1, 2, 5).Many cases of PMC may be self-limited if the offending agent is withdrawn; however, treatment with orally administered vancomycin or cholestyramine may be necessary. Vancomycin given in doses of 0.5 to 2.0 g per day has been shown to be effective in producing symptomatic improvement and a decrease in the titers of toxin in stool (5,8). The use of anion-exchange resins, such as cholestyramine, which bind the toxin appears to be efficacious in some cases, but the results are not as dramatic as those produced by vancomycin (5, 11).Several reports of relapse of PMC after oral vancomycin therapy have been noted and have led some authorities to suggest that the use of vancomycin plus cholestyramine or other antibiotics active against C. difficile might provide superior therapy (3, 6). However, potential difficulties arise with the combination since it has been demonstrated that vancomycin is approximately 90% bound by cholestyramine (4, 10).Although the presence of vancomycin does not inhibit binding of the cytotoxin by the resin, the concentration of vancomycin present is sharply reduced (4). These studies have examined the concentration of unbound vancomycin in the supernatant of centrifuged specimens.To our knowledge, the activity of bound van-comycin has not been examined. The purpose of this study was to examine the effects of cholestyramine binding of vancomycin in centrifuged and uncentrifuged samples. cholestyramine usually given within 24 h (2,000 mg and 12 g, respectively) were each mixed in 1 liter of the buffered saline. Five sets of mixtures were examined as follows: A, vancomycin (2 mg/ml); B, cholestyramine suspension (12 mg/ml); and C, D, and E, vancomycin (2 mg/ml) plus cholestyramine (12 mg/ ml). Each solution was tested in triplicate. All of the tubes were agitated in a water bath at 37°C for 45 min and then centrifuged for 15 min. Samples of the supernatant from A, B, and C were then immediately decanted and assayed for vancomycin activity. Tubes in D were blended with a Vortex mixer after centrifugation to resuspend the cholestyramine, and samples of the suspension were assayed ...