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Aim This was an in vitro study to analyse the susceptibility of Clostridium difficile isolates to rifampin and rifaximin. Methods Stool samples from patients who had nosocomial diarrhoea and C difficile toxin B at a university hospital between August 2006 and December 2007 were cultured for C difficile. Susceptibility of C difficile isolates to rifaximin and rifampin was determined by agar dilution and E strips, respectively. C difficile isolates were analysed via PCR for genes encoding toxins A and B, for binary toxin (BT), and for partial deletions of the tcdC gene (tcdC-del). Results Rifaximin exhibited high-level activity against 359 C difficile isolates, with MIC50 <0.01 µg/ml and MIC90 0.25 µg/ml; rifampin had MIC50 <0.002 µg/ml and MIC90 4 µg/ml. Among isolates analysed, 55 (15%) were positive for BT and tcdC-del. 28 (8% of 359) isolates were resistant to rifampin (≥32 µg/ml), of which 6 (2% of 359) were resistant to rifaximin and rifampin with MIC values ≥32 µg/ml. 2 of the 28 isolates resistant to rifampin were A+/B+/BT+/tcdC-del+, 5 were A+/B+/BT-/tcdC-del+, 4 were A+/B+/BT+/tcdC-del-, 13 were A+/B+/BT-/tcdC-del-, and 4 had no detectable toxin genes. Of the 11 isolates resistant to rifaximin alone, 1 was A+/B+/BT-/tcdC-del+, 2 were A+/B+/BT+/tcdC-del-, 6 were A+/B+/BT-/tcdC-del-, and 2 had no detectable toxin genes. Conclusions The study demonstrates that rifaximin has high-level activity against C difficile in vitro. Determination of resistance to rifampin by E strip did not predict rifaximin resistance. [ABSTRACT FROM AUTHOR]