Rabbit colonic mucosa was cultured in vitro in the presence of broad-spectrum antibiotics. DNA synthesis, as determined by [3H]thymidine incorporation into DNA, was inhibited by clindamycin (32%) and tetracycline (28%). Penicillin G, ampicillin, and metronidazole had no effect. When bile acids were added to the incubation at a concentration of 10 mxr, the more lipophilic drug, lincomycin, inhibited DNA synthesis by 38% (cholic acid) and by 23% (chenic acid). Bile acids alone did not alter thymidine incorporation. Inhibition by clindamycin demonstrated a gradual dose-response curve, with inhibition first noted at about 20 JLg of antibiotic/rnl and maximal inhibition at 1,000 µg/ml. The more biologically active metabolite of clindamycin, N-demethylclindamycin, inhibited DNA synthesis at concentrations of as low as 10 µg/ml. The inhibition was reversible, and the drugs did not affect thymidine uptake, thymidine pool concentrations, or protein or mucopolysaccharide synthesis at concentrations of up to 300 µg/ml. These data demonstrate a direct but reversible toxic action on mammalian colonic mucosa by certain antibiotics implicated in the production of pseudomembranous colitis and are consistent with the hypothesis that such a toxic action may be one factor in the pathogenesis of antibiotic-associated colitis.