AIM: To study the method of dissociation, culture and investigate its morphologic changes in vitro of interstitial cells of Cajal (ICC).METHODS: Enzymatic digestion and Ficoll density centrifugation were used to dissociate ICC from the ileal segment of mice. Factors including contamination, Ca2+, Mg2+ and collagenase, and stem cell factor, etc., were investigated.ACK2, the antibody of c-kit, was used to identify the cultured ICC. Both light microscope and fluorescence microscope were used to observe the changes of ICC in vitro.RESULTS: The method for dissociation and culture of ICC in vitro was successfully established. After 24 h, cultured ICC exhibited a few axis-cylinders, and longer axis-cylinders were observed to form synapse of each other after 3 d. More widespread connections formed within 7 d in vitro. The changes of its morphologic character were obvious within 7 d; however, there were no obvious morphologic changes after 30 d.CONCLUSION: Many factors can influence the dissociation and culture of ICC.