Purpose Patients with adenocarcinoma of unknown primary origin (ACUP) constitute approximately 4% of all malignancies. For effective treatment of these patients, it is considered optimal to identify the primary tumor origins. Currently, the success rate of the diagnostic work-up is only 20% to 30%. Our goal was to evaluate the contribution of gene expression profiling for routine clinical practice in patients with ACUP. Patients and Methods Formalin-fixed, paraffin-embedded (FFPE) samples were obtained from 84 patients with a known primary adenocarcinoma and from 38 patients with ACUP. An extensive immunohistochemical panel classified 16 of the patients with ACUP, whereas 22 patients remained unclassified for their histogenetic origin. Information about staging procedures and clinical follow-up were available in all patient cases. The expression data were analyzed in relation to clinicopathologic variables and immunohistochemical results. Results The gene expression–based assay classified the primary site correctly in 70 (83%) of 84 patient cases of primary and metastatic tumors of known origin, with good sensitivity for the majority of the tumor classes and relatively poor sensitivity for primary lung adenocarcinoma. Gene expression profiling identified 15 (94%) of 16 patients with initial ACUP who were classified by immunohistochemistry, and it made a valuable contribution to a potential site of origin in 14 of the 22 patients with ACUP. Conclusion The gene expression platform can classify correctly from FFPE samples the majority of tumors classes both in patients with known primary and in patients with ACUP. Therefore, gene expression profiling represents an additional analytic approach to assist with the histogenetic diagnosis of patients with ACUP.