This study investigates whether CD44 immunopositive cells truly correspond to the interstitial cells of Cajal (ICCs) in the muscular layers of the gastrointestinal tract in guinea pigs and Balb/c mice using immunohistochemistry with antibodies directed against CD44, Kit, vimentin and neurofilament 200 (NF200). All the sub-groups of ICCs were immunopositive for the anti-Kit antibody in the muscular layers of stomach, small intestine and colon in both cross sections and whole-mount preparations. Kit/CD44/vimentin triple immunolabeling showed that all the ICCs in different segments and muscular layers of the digestive tract were CD44, Kit and vimentin immunopositive. Kit/CD44/NF200 triple immunolabeling revealed that neither enteric nerves nor other major cells were CD44 immunopositive in the muscular layers, apart from the ICCs. CD44 and Kit were co-localized in the same group of cells, apart from a very small number (0.6%) of CD44 immunopositive cells that were not Kit immunopositive. Our results indicate that these CD44 immunopositive cells truly correspond to ICCs, thus immunolocalisation of CD44 can be used as a special marker, in addition to Kit, to identify ICCs in the digestive tract in adult guinea pigs and mice.