The tumor-suppressor protein Dpc4 (Smad4, Madh4) regulates gene expression. On binding of an extracellular ligand of the extensive transforming growth factor (TGF) superfamily to its cognate receptor complex, latent cytoplasmic Dpc4 is activated and translocated into the nucleus to function as part of various DNA-binding transcriptional activator complexes. The most relevant ligand/receptor pair to control the tumor suppressive function of Dpc4 remains uncertain, but is usually assumed to be TGF-beta and its heteromeric receptor. We exploited a fortuitous experiment of nature to directly test this hypothesis: the TGF-beta type II receptor gene is inactivated by mutation in nearly all colorectal carcinomas having microsatellite instability, as seen in hereditary nonpolyposis colorectal cancer (HNPCC) and in sporadic medullary colorectal cancers. Using a specific and sensitive immunohistochemical label for Dpc4, we examined nuclear localization of Dpc4 in 13 HNPCC, six medullary, and 41 sporadic nonmedullary colorectal carcinomas. In agreement with published rates, two (5%) of 41 sporadic tumors showed complete loss of Dpc4 protein, indicative of genetic inactivation. All 13 HNPCC and six medullary tumors had intact cytoplasmic and nuclear Dpc4 localization. The TGFBR2 gene was sequenced in three of the cancers from patients with HNPCC, and all of these harbored inactivating mutations. The specificity of the immunohistochemical assay was demonstrated in xenograft tumors of syngeneic cell lines that differed in DPC4 genetic status because of an engineered gene knockout. Thus, nuclear localization of Dpc4 can be maintained in cells with inactivated TGF-beta type II receptors, suggesting the persistence of tumor-suppressive action of an upstream signaling input, most likely a ligand/receptor complex distinct from TGF-beta. Identification of the relevant input would be expected to have implications for the understanding of tumorigenesis and the design of rational biological therapy.