Clostridium difficile intestinal infection is a major nosocomial hazard in patients receiving antimicrobial therapy. Rationale for rapid diagnosis include lifesaving antimicrobial therapy in patients with severe colitis and early isolation measures for transmission control. We have therefore analysed the sensitivity, specificity and predictive value of GLC identification of isocaproic acid in diarrheic stools from adult hospitalized patients in comparison with selective fecal culture on Cycloserine Cefoxitin Fructose Agar. During the study period, the prevalence of positive culture for C. difficile was 38/595 fecal specimens (6.4%). Compared with culture, GLC had a sensitivity of 24/38 (63%) and a specificity of 524/557 (94%). The predictive value of a positive GLC was 24/57 (42%) and of a negative GLC was 524/538 (97%). Measurement of the isocaproic acid peak height did not allow determination of a cutt-off value improving the test accuracy. The sensitivity of detection of isocaproic acid in stools by GLC is too low to be used as screening test for C. difficile infection. However, in a low prevalence population, a positive GLC test increased the pre-test probability of infection sevenfold.