Two monoclonal antibodies (MAbs) AUAI and HMFGI recognize antigens located on different membrane domains of polarized epithelial cells. We have assessed the accessibility of these antigens in multicellular tumour spheroids produced in culture using a well‐polarized (HRA‐19) and a non‐polarized cell line (LoVo) of human large‐bowel carcinoma origin. Multicellular spheroids of HRA‐19 cells develop polarity, so that the membrane which is in contact with the culture medium (apical) becomes antigenically distinct from the membrane facing the centre of the spheroids (basolateral). This was confirmed by immunostaining sections of spheroids with 2 MAbs, AUAI and HMFG I. AUA I recognizes an antigen located exclusively on the basolateral membranes of polarized epithelial cells, and stained only internal membranes in spheroid sections. Conversely HMFG1, which recognizes an antigen located on the apical membranes, stained only the periphery of the spheroids. These 2 MAbs were then radiolabeled with 125l and incubated with live spheroids for 4 hr at 37°C. Autoradiography of spheroid sections showed a marked difference between the 2 MAbs. 125l‐HMFGI‐radioantibody localized exclusively on the spheroid surface in a pattern identical to the in vitro immunostaining pattern, while 125l‐AUAI radioanti‐body showed no binding in spite of the uniform presence of antigen on all tumour cells basolaterally. This appeared to be the result of the inaccessibility of basolateral antigenic sites in well‐polarized epithelial cells because of the tight junctions connecting these cells at their apicai surfaces. In contrast to the HRA‐19 cell line LoVo, spheroids do not develop polarity; as a result, when stained with AUAI, variable antigenic expression all over the cell surface was seen. Autoradiographs of these spheroids showed 125I‐AUAI binding with a penetration to a depth of about 1–3 cells, while HMFG I which shows no reactivity with this cell line in vitro, did not bind. This phenomenon was further investigated in xenografts of the HRA‐19 cell line. It was shown that in a well‐differentiated adenocarcinoma where the tumour cells forming acini are arranged in a polarized fashion, the luminal antigenic sites may be inaccessible to the injected MAb. The striking differences in binding of MAbs on polarized and unpolarized tumours indicate the importance of cell polarization and exact location of antigenic sites for in vivo immunotargeting.