Anti‐serum raised against the human cholecystokinin B (CCKB)/gastrin receptor was used in Western blotting to differentiate the cellular locations of receptor isoforms expressed by human gastro‐intestinal (GI) tumour cell lines. Using anti‐serum directed against the amino‐terminal extracellular tail of the CCKB/gastrin receptor, 8/9 cell lines were shown to express immunoreactive proteins of either m.w. 70 or 40 kDa, or both. Both isoforms were found to be associated with intracellular, non‐nuclear membranes, whereas only the 70 kDa protein was expressed in the plasma membrane. Receptor expression was related to gastrin production and secretion. Both progastrin and glycine‐extended gastrin‐17 were produced and secreted by the tumour cell lines; however, carboxy amidated gastrin was not detected by radioimmunoassay. A CCKB/gastrin receptor transfectant NIH3T3 cell line did not produce detectable gastrin and showed exclusive expression of the 70 kDa receptor on the plasma membrane. One cell line had <50 pg/ml cell‐associated progastrin and no detectable receptor form. Cell lines expressing 50–150 pg/ml had both 40 and 70 kDa receptor forms. Those expressing >150 pg/ml progastrin had only the 40 kDa isoform, which was shown to be exclusively expressed on intracellular, non‐nuclear membranes, in one of the cell lines. Of the 4 cell lines exclusively expressing the lower m.w. receptor, 3 had gastrin present within the cell, which was not secreted. Thus, if cell‐ associated gastrin induces a proliferative effect, it may be by an intracrine pathway. Our study has identified the presence of CCKB/gastrin receptor isoforms in different cellular locations and may help toward understanding the complex autocrine and intracrine pathways mediated by gastrin peptides. Int. J. Cancer 77:572–577, 1998. © 1998 Wiley‐Liss, Inc.