To study the role of cell-surface expression of a tumorselective heat-shock protein 70 (Hsp70) in vivo, the coloncarcinoma cell line CX2, and the clonal sub-lines CX ؉ and CX ؊ , which differ in Hsp70 cell-surface expression, but not in MHC and adhesion-molecule expression, were implanted into immunodeficient SCID/beige mice by s.c., i.p., i.v. and orthotopic (o.t.) inoculation. On day 18 after s.c. injection, all animals developed s.c. tumors, ranging in size from 2.5 to 3 cm 2 . Phenotypic characterization of single-cell suspensions generated from freshly isolated tumor material revealed that the pattern of cell-surface expression is identical to that of the injected tumor cells from cell culture. Comparable results were obtained following i.p. inoculation of CX ؉ and CX ؊ cells. Macroscopic and microscopic evaluation of lymph nodes, lung, liver and spleen at autopsy of tumor-bearing mice showed no tumor burden except the primary tumor, following s.c. or i.p. injection. After i.v. inoculation of CX ؉ and of CX ؊ cells, weak tumor growth was observed in lung and liver, the Hsp70 cell-surface-expression pattern on these tumors being identical to that of the injected cells. However, o.t. injection of colon-carcinoma cell lines CX ؉ and CX ؊ into the cecum resulted in tumor growth at the injection site and in spread of distant metastases in lung, liver and spleen. Most interestingly, and in contrast to the primary colon carcinomas, metastases of CX ؉ and of CX ؊ tumor cells both revealed strong Hsp70 plasma-membrane expression, although the total amount of cytoplasmic Hsp70 was comparable.