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In advanced prostate cancer, there is a need for biomarkers to monitor response to therapy and determine prognosis. Current tests for circulating tumor cells (CTCs) rely on epithelial markers with limited sensitivity and specificity. We showed that the staining pattern of nucleolin, a protein associated with proliferative cells, aids in the classification of prostate cancer CTCs.Circulating tumor cells (CTCs) have great potential as circulating biomarkers for solid malignancies. Currently available assays for CTC detection rely on epithelial markers with somewhat limited sensitivity and specificity. We found that the staining pattern of nucleolin, a common nucleolar protein in proliferative cells, separates CTCs from white blood cells (WBCs) in men with metastatic prostate cancer.Whole peripheral blood from 3 men with metastatic prostate cancer was processed with the AccuCyte CTC system (RareCyte, Seattle, WA). Slides were immunostained with 4′,6-diamidino-2-phenylindole (DAPI), anti–pan-cytokeratin, anti-CD45/CD66b/CD11b/CD14/CD34, and anti-nucleolin antibodies and detected using the CyteFinder system. DAPI nucleolin colocalization and staining pattern wavelet entropy were measured with novel image analysis software.A total of 33,718 DAPI-positive cells were analyzed with the novel imaging software, of which 45 (0.13%) were known CTCs based on the established AccuCyte system criteria. Nucleolin staining pattern for segmentable CTCs demonstrated greater wavelet entropy than that of WBCs (median wavelet entropy, 6.86 × 107 and 3.03 × 106, respectively; P = 2.92 × 10−22; approximated z statistic = 9.63). Additionally, the total nucleolin staining of CTCs was greater than that of WBCs (median total pixel intensity, 1.20 × 105 and 2.55 × 104 integrated pixel units, respectively; P = 2.40 × 10−21; approximated z statistic = 9.41).Prostate cancer CTCs displayed unique nucleolin expression and localization compared to WBCs. This finding has the potential to serve as the basis for a sensitive and specific CTC detection method.