We compared the activities of ciprofloxacin and levofloxacin with those of the newer fluoroquinolones grepafloxacin, moxifloxacin, sparfloxacin and trovafloxacin against Clostridium difficile isolates. As there is good evidence of marked clonal spread of C. difficile, we studied both genotypically distinct (n = 26) and indistinguishable (n = 28) isolates as determined by random amplified polymorphic DNA and ribosomal spacer PCR fingerprinting. The indistinguishable strains examined represent the main UK epidemic C. difficile clone. For 17 of 54 strains (31%) we were unable to read MICs following inocula preparation using Mueller-Hinton broth. Using Schaedler's broth for inocula preparation 93% of strains had readable MICs, although geometric mean MICs were uniformly higher (2.5- to 5.4-fold) compared with results using Mueller-Hinton broth. Moxifloxacin and trovafloxacin, followed by grepafloxacin, were the most active fluoroquinolones tested and were 3- to 4-fold more active than older agents such as ciprofloxacin by both MIC methods. Unexpectedly, clonal C. difficile strains had markedly reduced susceptibility compared with the distinct strains to each of the fluoroquinolones tested. Clonal strains were more than seven-fold or 12- to 29-fold less susceptible (according to geometric mean MICs) than distinct strains to both moxifloxacin and trovafloxacin, depending on the MIC method used. It remains to be seen whether the enhanced activity of new fluoroquinolones such as moxifloxacin in comparison with other fluoroquinolones against C. difficile implies that these agents are unlikely to be associated with C. difficile infection. However, clinical use of new generation fluoroquinolones in elderly hospitalized patients where C. difficile is endemic requires further study, particularly given the reduced antibiotic susceptibility to all fluoroquinolones of the readily transmissible UK C. difficile clone.