Purpose: There have been recent reports of a rising incidence of C. difficile-associated diarrhea (CDAD) as well as more virulent strains of the organism. This information, in combination with the limitations of existing treatments (side effects, resistance, cost and recurrence), has prompted studies of new therapies. Rifaximin is a derivative of rifamycin newly available in the U.S. and is characterized by broad-spectrum anti-microbial activity, minimal systemic absorption (<0.4%), an excellent safety profile, and animal data showing efficacy against C. difficile. We performed an investigator-initiated, prospective, open-label trial to assess the efficacy and safety of rifaximin 400 mg PO TID as first-line therapy in patients with CDAD. Methods: Eligible patients were those with >3 unformed stools/day, enzyme immunoassay (EIA)-positive for C.difficile toxin and who had not been treated with metronidazole or vancomycin for at least 2 days before the positive EIA (treatment-naïve). Patients received study drug and were asked to maintain a symptom diary. The primary efficacy outcome was symptom resolution; secondary assessments were time to last unformed stool (TLUS), EIA for C. difficile toxin at day 10, and symptomatic relapse 2 weeks after treatment. In addition, drug-related adverse events (AEs) were recorded. Results: 8 patients (mean age 55y, SD ± 15y, 4M, 4F) with CDAD were recruited and received 10 days of rifaximin. All 8 patients reported symptom resolution with treatment and 7 reported a median TLUS of 127.5h (range 84–187h). The 8th patient (with a history of AML and prior CDAD) had 3 days without a bowel movement but then during treatment reported pasty stool and was treated for persistent CDAD after day 10. On day 10, 4 patients had a negative EIA for C. difficile toxin, 1 patient had persistent positive EIA but was asymptomatic, and 2 asymptomatic patients did not submit samples. So far, 5 of 7 patients (including the positive EIA patient) have completed 2 week follow-up and there have been no relapses. There were no drug-related AEs in this study. Conclusions: This open-label study demonstrates that rifaximin 400 mg PO TID is an effective first-line therapy for the treatment of CDAD and has an excellent safety profile. In addition, these results suggest that this therapy may not have significant relapses. Further study of this promising agent in patients with CDAD is warranted.