Fidaxomicin has recently been approved for the treatment of Clostridium difficile infection (CDI). As part of phase III studies, plasma and fecal samples were analyzed for concentrations of fidaxomicin and its metabolite, OP-1118. Plasma samples were collected before and after dose receipt on the first and last days of therapy, and fecal samples were collected on the last day of therapy. Samples were analyzed for fidaxomicin and OP-1118 (metabolite), using validated liquid chromatography/tandem mass spectrometric methods. Plasma concentrations were low for both fidaxomicin (mean [± standard deviation {SD}], 22.8 ± 26.7 ng/mL and 28.5 ± 33.4 ng/mL on the first and last days of therapy, respectively) and OP-1118 (mean [± SD], 44.5 ± 50.4 ng/mL and 85.6 ± 131 ng/mL, respectively). In contrast, fecal levels were >1000 µg/g for fidaxomicin and >800 µg/g for OP-1118. Fidaxomicin mean fecal levels were >5000 times the minimum inhibitory concentration for C. difficile of 0.25 µg/mL.