Background: Small intestinal bacterial overgrowth (SIBO) has been proposed to be common in irritable bowel syndrome (IBS), with altered small-bowel motility as a possible predisposing factor. Aim: To assess the prevalence of SIBO, by culture of small-bowel aspirate, and its correlation to symptoms and motility in IBS. Methods: 162 patients with IBS who underwent small-bowel manometry and culture of jejunal aspirate were included. Cultures from 26 healthy subjects served as controls. Two definitions of altered flora were used: the standard definition of SIBO (≥ 105 colonic bacteria/ml), and mildly increased counts of small-bowel bacteria (≥ 95th centile in controls). Results: SIBO (as per standard definition) was found in 4% of both patients and controls. Signs of enteric dysmotility were seen in 86% of patients with SIBO and in 39% of patients without SIBO (p = 0.02). Patients with SIBO had fewer phase III activities (activity fronts) than patients without SIBO (p = 0.08), but otherwise no differences in motility parameters were seen. Mildly increased bacterial counts (≥5×103/ml) were more common in patients with IBS than in controls (43% vs 12%; p = 0.002), but this was unrelated to small intestinal motility. No correlation between bacterial alterations and symptom pattern was observed. Conclusions: The data do not support an important role for SIBO according to commonly used clinical definitions, in IBS. However, mildly increased counts of small-bowel bacteria seem to be more common in IBS, and needs further investigation. Motility alterations could not reliably predict altered small-bowel bacterial flora.