Based on the hypothesis that intestinal clostridia play a role in late-onset autism, we have been characterizing clostridia from stools of autistic and control children. We applied the TaqMan real-time PCR procedure to detect and quantitate three Clostridium clusters and one Clostridium species, C. bolteae, in stool specimens. Group-and species-specific primers targeting the 16S rRNA genes were designed, and specificity of the primers was confirmed with DNA from related bacterial strains. In this procedure, a linear relationship exists between the threshold cycle (C T ) fluorescence value and the number of bacterial cells (CFU). The assay showed high sensitivity: as few as 2 cells of members of cluster I, 6 cells of cluster XI, 4 cells of cluster XIVab, and 0.6 cell of C. bolteae could be detected per PCR. Analysis of the real-time PCR data indicated that the cell count differences between autistic and control children for C. bolteae and the following Clostridium groups were statistically significant: mean counts of C. bolteae and clusters I and XI in autistic children were 46-fold (P ؍ 0.01), 9.0-fold (P ؍ 0.014), and 3.5-fold (P ؍ 0.004) greater than those in control children, respectively, but not for cluster XIVab (2.6 ؋ 10 8 CFU/g in autistic children and 4.8 ؋ 10 8 CFU/g in controls; respectively). More subjects need to be studied. The assay is a rapid and reliable method, and it should have great potential for quantitation of other bacteria in the intestinal tract.Autism is a complex disease with unclear causes. Many autistic subjects exhibit a range of gut disorders, which include constipation, diarrhea, retention of gas, and abdominal pain and discomfort. Abnormal gut microflora may play a role in these problems. Research into the characteristics of the gut flora in autism has been limited. In our initial studies that characterized the fecal bacterial composition by culturing, we noted abnormalities in the fecal bacterial composition of children with autism compared to age-and sex-matched controls. We found higher counts of clostridia overall and more species of clostridia in stools of autistic children than in healthy children (11). In particular, Clostridium bolteae, a novel species that we described previously (29; called Clostridium clostridioforme in reference 11), caught our attention because it was cultured from 5 of 15 autistic children, but none of 8 controls. However, it is well known that traditional culture-based methods, while very important, result in a significant underestimation of bacteria present in fecal samples (14,19,30).Molecular techniques introduced in microbial ecology have made it possible to study the composition of intestinal flora in a culture-independent way based on the detection of rRNA genes. Although these methods, such as fluorescent in situ hybridization (12,13,19), denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis (8,10,28), and the 16S rRNA gene clone library method (15,27,30), have been applied successfully for studying the eco...