Objective: To investigate the method of in vitro isolation, culture and identification of interstitial cells of Cajal (ICC) from the small intestine of rats. Methods: Neonatal SD rats at 5-10 d after birth were sacrificed by cervical dislocation, and their small intestinal segments were excised under sterile conditions. Under a dissecting microscope, the layers of smooth muscle of the small bowel were cut into small pieces after the mesentery, mucosal and submucosal layers were carefully stripped off, and then the tissue pieces were cultured in the DMEM medium containing stem cell factor. After that, cells emanating around the tissue blocks and their morphological characteristics were successively observed with an inverted microscope, and the cell phenotype was identified by immunofluorescence staining using specific antibody against receptor tyrosine kinase c-kit. Results: One week after culture, cells were found growing from the tissue blocks under inverted microscope, which took on a fusiform or triangular shape with several short processes. As the length of culture period increased, the cellular processes were gradually elongated and interconnected with those from other cells, forming a net-work structure. These cells were positive for immunofluorescence staining of c-kit antibody. Conclusion: This study has successfully established a method for the primary culture of ICC from the small intestine of rats in vitro, which may provide a basis for research into the biological function of ICC and the relationship between the ICC and gastrointestinal motility disorders.