Interstitial cells of Cajal (ICC) have recently been found to display phenotypic changes. The present study is designed to determine whether phenotypic changes occur in ICC associated with an inflammatory microenvironment and whether the ICC phenotype could be recovered after the discontinuation of inflammatory stimuli. Immunohistochemistry studies revealed that the functional ICC marker, c-kit, was markedly reduced in patients with Hirschsprung's disease (n = 34) compared with controls (n = 12), whereas another marker of ICC, CD34, was not altered significantly. Compared with the vehicle group (n = 6), intraperitoneal injection of lipopolysaccharide (LPS; 1.5 mg/kg) in mice (n = 6) significantly induced plasma tumor necrosis factor-alpha (TNF-alpha) levels as determined by enzyme-linked immunosorbent assay. Western blot and real-time polymerase chain reaction assessment further showed that LPS injection markedly suppressed intestinal c-kit protein and mRNA expression, which could be blocked by Toll-like receptor 4 (TLR4) deficiency (n = 6) rather than TLR2 deficiency (n = 6) and had no effects on CD34. Compared with the vehicle group (n = 6), intraperitoneal TNF-alpha (30 mu g/kg) administration (n = 6) also significantly reduced intestinal c-kit protein and mRNA levels but not CD34 levels. However, the reduction of c-kit induced by TNF-alpha injection was not suppressed by TLR4 deficiency (n = 6). Intestinal c-kit protein and mRNA levels were markedly restored after the discontinuation of TNF-alpha administration for 7 days. Moreover, immunofluorescence analysis of primary ICC further confirmed that exposure to TNF-alpha for 24 h suppressed c-kit expression, which could be restored after discontinuation of TNF-alpha exposure. CD34 expression was not altered upon exposure to TNF-alpha. Thus, phenotypic changes in ICC occur in an inflammatory microenvironment in the gut and LPS, TLR4 and TNF alpha are crucial to this process.