Localization and production of α2-macroglobulin (α2M), a multifunctional binding protein with protease and cytokine scavenging properties, was studied in situ in rat livers containing experimentally induced colon carcinoma metastases by means of immunocytochemistry and in situ hybridization methods. The study was performed to investigate whether α2M production by hepatocytes plays a role in the defense against the growth of metastases on the basis of its protease inhibiting capacity. It was found that colon cancer cells in all developmental stages of the metastases contained large amounts of messenger RNA (mRNA) of α2M but hardly any α2M protein. Cancer cells in culture contained large amounts of both mRNA and protein of α2M. In contrast, stromal cells and liver cells did not show positivity for α2M mRNA above background levels. The exception was a few layers of hepatocytes around the latest stage of metastases. Hepatocytes contained both α2M mRNA and protein only when Kupffer cells were present, indicating that α2M mRNA production was induced via Kupffer cells. On the other hand, α2M protein was found in high amounts in the sinusoids and stroma of all metastases, irrespective of their developmental stage. Increased levels of α2M could not be detected in serum in all but one rat tested (n=8). It is concluded that production of α2M by hepatocytes occurs only around the latest developmental stage of metastases and that α2M does not play a significant role in the defense against metastatic cancer growth in rat liver. In contrast, cancer cells produce and secrete large amounts of α2M, which seems to be linked with their tumorigenicity. We suggest that this α2M captures cytokines rather than proteases by complex formation. These complexes were observed using immunocytochemical staining for α2M protein indicating that it was captured by either stromal cells, sinusoidal cells, or hepatocytes that are in direct contact with cancer cells, Therefore, changes in serum levels of α2M were limited, indicating that these levels do not reflect local production and effects of α2M.