Background: Molecular tumor profiling has potential importance in identifying the tissue of origin in patients with cancer of unknown primary (CUP). We retrospectively performed the Tissue of Origin test, an FDA-cleared commercially available gene microarray assay, on biopsy specimens from patients with CUP. Assay results were correlated with clinical and pathologic features, and with previous results using the Veridex 10-gene CUP assay, a molecular RT-PCR assay designed to detect 6 primary sites. Methods: Archival formalin-fixed, paraffin-embedded biopsy specimens from 48 patients with CUP were tested. The assay results were reported without incorporation of clinicopathologic information except biopsy site and patient gender. The assay results were correlated with clinicopathologic information, treatment results, and with results of the previously performed Veridex assay. Results: The Tissue of Origin test was successfully performed in 45 tumor specimens. In 43 of 45 assays (96%), a specific tissue of origin was predicted. The most commonly identified tissues of origin included: lung (11), pancreas (6), sarcoma (6), ovary (5), and colon (4). Most diagnoses were compatible with the clinical features, IHC staining, and response to treatment. The finding of 6 sarcomas was unusual in this patient population and was suggested by routine pathology in only 1 patient. The Tissue of Origin test provided predictions in a higher percentage of patients than did the Veridex CUP assay (96% versus 53%). However, concordance between assay results was relatively low. Conclusions: The Tissue of Origin test provided predictions of the primary site in 96% of patients with CUP. Predictions were generally consistent with clinicopathologic features. Agreement between the Tissue of Origin test and the Veridex CUP assay was relatively low, possibly related to the limited number of genes assessed by the Veridex CUP assay. Additional trials are necessary to confirm the value of these assays in patient management.