The PRO Disc (Carr-Scarborough Microbiologicals, Inc., Decatur, Ga.) can be used to screen for L-proline-aminopeptidase produced by Clostridium difficile grown on cycloserine-cefoxitin-fructose agar (CCFA). Fifty stored isolates of C. difficile (48 toxin-positive and 2 toxin-negative isolates) and 47 fresh C. difficile isolates (39 toxin-positive and 8 toxin-negative isolates) were all PRO Disc positive. Other Clostridium species that were PRO Disc positive could be differentiated from C. difficile by failure to grow on CCFA, different colonial morphology on CCFA, or morphology upon Gram staining.