Non-toxigenic strains of Clostridium difficile are highly effective in preventing toxigenic C. difficile infection in hamsters when given following a single dose of an antimicrobial agent. The goal of this study was to determine the ability of non-toxigenic C. difficile to colonise hamsters during administration of an antibiotic to which the organisms are resistant - ceftriaxone - and an antibiotic to which they are susceptible - ampicillin - and to determine if non-toxigenic colonisation is protective against toxigenic strain challenge. Groups of four or five hamsters were administered daily ceftriaxone 60 mg/kg/d intraperitoneally or ampicillin 60 mg/kg/d orally for 5 days. Three non-toxigenic strains of C. difficile, M3, M23, and T7 (MICs 96-128 mg/L) were each given orally at a dose of 1 x 10(6) spores to groups of five animals 3h after the first dose of ceftriaxone. All animals were colonised successfully by day 3 of the study and when challenged with 1 x 10(6) spores of toxigenic strain J9 (MIC >256 mg/L) on day 3 all animals survived, whereas the control animal given ceftriaxone, but not non-toxigenic C. difficile, died within 48h of challenge. When groups of four hamsters were given ampicillin, administration of non-toxigenic strain M3 (MIC 2 mg/L) or toxigenic strain J9 (MIC 0.75 mg/L) at 1 x 10(6) spores did not result in any colonisation or infection of the animals until day 8, 3 days after the last ampicillin dose. A protection study was designed by giving M3 spores to groups of five animals daily for 5 days beginning on day 1, 3, or 5 of ampicillin. Toxigenic challenge was given with J9 spores on day 3 of each M3 regimen. M3 colonised all animals by day 8 and none became infected with J9. Colonisation by non-toxigenic C. difficile is an effective prevention strategy during antibiotic administration of ceftriaxone or ampicillin, but multiple-day administration is required for ampicillin and colonisation does not occur until several days after the drug is discontinued.