The aim of this study was to assess the presence of interstitial Cajal-like cells (ICLCs) in the human gallbladder and to determine their distinctive characteristics on the basis of double immunohistochemical staining. Gallbladder specimens were obtained from 30 patients subjected to elective laparoscopic cholecystectomy for symptomatic gallstone disease. Tissue samples were fixed in 4% phosphate-buffered paraformaldehyde, processed, embedded in paraffin, and, after sectioning, routinely stained with HE. Tissue antigens were retrieved using the heat-induced epitope retrieval (HIER) method. For simultaneous visualisation of two antigens, an indirect double immunofluorescence procedure was applied. ICLCs were defined as CD117-immunopositive and tryptase-immunonegative objects. They were predominantly fusiform in shape with sparse branches that were visible in some sections. ICLCs were observed throughout the organ including the gallbladder's fundus, body (corpus) and neck, being most numerous in the corpus. The ICLCs were detected almost exclusively within the muscularis propria and they were arranged parallel to smooth muscle cells. The following subpopulations of ICLCs were observed: ICLC-IM (intramuscular ICLCs) localised between smooth muscle fibres forming one muscle bundle; and ICLC-IB (interbundle ICLCs) localised within the connective tissue separating smooth muscle bundles. Thus, the presence of ICLCs in the human gallbladder was clearly identified, demonstrated by double immunohistochemistry which was found to be a reliable method for differentiating ICLCs from mast cells.