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Intestinal mucosal pathology, characterized by ileo-colonic lymphoid nodular hyperplasia (LNH) and mild acute and chronic inflammation of the colorectum, small bowel and stomach, has been reported in children with autistic spectrum disorder (ASD).To assess ileo-colonic LNH in ASD and control children and to test the hypothesis that there is an association between ileo-colonic LNH and ASD in children.One hundred and forty-eight consecutive children with ASD (median age 6 years; range 2–16; 127 male) with gastrointestinal symptoms were investigated by ileo-colonoscopy. Macroscopic and histological features were scored and compared with 30 developmentally normal (non-inflammatory bowel disease, non-coeliac disease) controls (median age 7 years; range 1–11; 25 male) showing mild non-specific colitis in 16 cases (13 male) and normal colonic histology in 14 cases (12 male). Seventy-four ASD children and 23 controls also underwent upper gastrointestinal endoscopy. The influence on ileal LNH of dietary restriction, age at colonoscopy, and co-existent LNH elsewhere in the intestine, was examined.The prevalence of LNH was significantly greater in ASD children compared with controls in the ileum (129/144 (90%) vs. 8/27 (30%), P<0.0001) and colon (88/148 (59%) vs. 7/30 (23%), P=0.0003), whether or not controls had co-existent colonic inflammation. The severity of ileal LNH was significantly greater in ASD children compared with controls, with moderate to severe ileal LNH present in 98 of 144 (68%) ASD children versus 4 of 27 (15%) controls (P<0.0001). Severe ileal LNH was associated with co-existent colonic LNH in ASD children (P=0.01). The presence and severity of ileal LNH was not influenced by either diet or age at colonoscopy (P=0.2). Isolated ileal LNH without evidence of pathology elsewhere in the intestine was a rare event, occurring in less than 3% of children overall. On histopathological examination, hyperplastic lymphoid follicles are significantly more prevalent in the ileum of ASD children (84/138; 61%) compared with controls (2/23; 9%, P=0.0001).Ileo-colonic LNH is a characteristic pathological finding in children with ASD and gastrointestinal symptoms, and is associated with mucosal inflammation. Differences in age at colonoscopy and diet do not account for these changes. The data support the hypothesis that LNH is a significant pathological finding in ASD children.