For the clinical diagnosis and appropriate therapy of patients with cancer of unknown primary (CUP), biopsies of the tumor metastases are generally examined histopathologically and by immunohistochemistry (IHC). Gene expression profiling (GEP) is a new diagnostic technique that might further contribute to tumor specification. Biopsies of 43 CUP cases underwent a retrospective central histopathological and immunohistochemical review and centrally performed GEP using CupPrint(TM). Two samples could not be evaluated by IHC due to small biopsy size or loss of immunoreactivity. One of these and 17 other samples were not suited for GEP due to RNA degradation. In 13 out of the remaining 24 cases (54%), the same primary was proposed by IHC and GEP and was supported by the clinical findings, furthermore leading to the doubtless identification of the primary in four out of these. In seven cases, there was discordance between IHC and GEP, with the clinical picture being more in line with IHC in three and with GEP in four cases. Four cases had to remain undecided because the primary tumors suggested by IHC and GEP were not supported. In conclusion, overlap between IHC and GEP results and the clinical presentation was noted in the majority of those true CUP cases that were evaluable with both techniques. Therefore, GEP can be a complementary diagnostic technique assisting immunohistochemical profiling of cancer biopsies with unknown primary.