The reliability of a single jejunal culture in the diagnosis of small bowel bacterial overgrowth has recently been questioned. Seventy-seven patients thought to have bacterial overgrowth, defined as a jejunal culture yielding at least 10(6) organisms per milliliter of aspirate, took part in the study. Bacterial overgrowth was found in 74% of the patients with predisposing conditions and in 32% of those with no clear causes of bacterial colonization. The intestinal juice of some patients was taken at two different levels of the proximal jejunum, using both the closed- and open-tube systems. Highly significant correlations (rs = 0.90, p less than 0.001) were found between the numbers of bacteria per milliliter at the 2 jejunal levels and between the numbers of bacteria per milliliter of jejunal aspirate obtained from the closed and open tubes (rs = 0.84, p less than 0.001). Compared with the jejunal culture, the gas chromatography of volatile fatty acids in jejunal aspirate and the glucose- and lactulose-hydrogen breath tests showed sensitivities of 56%, 62%, and 68% and specificities of 100%, 83%, and 44%, respectively. This work demonstrates the reliability of jejunal cultures and the inadequacy of breath hydrogen testing in the prediction of positive jejunal cultures. When results of testing for volatile fatty acids in jejunal aspirates are positive, this always indicates the presence of bacterial overgrowth; thus, this procedure would avoid the more complicated, time-consuming, and costly bacteriological analysis of jejunal samples.