The ultrastructure of the interstitial cells of Cajal (ICC) has been examined in birds, but the distribution of these cells remains obscure because a suitable marker is lacking. In the present study, the identification and expression of c-Kit-positive cells in the chicken intestine were demonstrated by means of in situ hybridization histochemistry and the expression of the c-Kit gene by real-time quantitative PCR. Two types of cells stained positive for c-Kit mRNA. The first group consisted of spindle-shaped or bipolar cells identified as ICC. The ICC were found at a variety of locations: at the level of the myenteric plexus between the circular and longitudinal muscle and intermingled with smooth muscle cells within muscle bundles in the circular and longitudinal muscle layers. The ICC were also identified along the submucosal layer. The second group was composed of round-shaped cells, which resembled mast cells. Mast cells were mainly found in the lamina propria region as well as in the submucosal layer. The expression of the c-Kit gene by real-time quantitative PCR revealed the expression of c-Kit mRNA throughout the lamina muscularis and mucosa of the intestine; however, the quantitation was variable in different regions. This study reveals conclusively for the first time the distribution of ICC, quantifies the expression of c-Kit mRNA in the intestine of adult chicken, and also compares the c-Kit-positive cell types morphologically.