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Abstract Background In this study we investigated the expression of vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR) and prostate-specific membrane antigen (PSMA) to analyze their potency as targets for the detection of lymph node (LN) metastases of urothelial carcinoma of the bladder. Methods Antigen expression was determined in 40 samples with urothelial carcinoma and compared to 17 matched samples without metastases by immunohistochemistry. The total immunostaining score (TIS 0–12) was determined as the product of a proportion score (PS 0–4) and intensity score (IS 0–3). Results VEGF expression was high in primary tumor and LN metastases (median TIS 8 in both) and VEGF expression was also seen in LNs without metastases (median TIS 6). EGFR expression was low in primary tumor and LN metastases (median TIS 3 and 2 respectively) and absent in LNs without metastases. PSMA expression was low in samples with urothelial carcinoma (median TIS 2). Conclusion VEGF shows moderate to high expression levels in both primary tumors and LN metastases and could be a candidate as a target agent for imaging modalities of urothelial carcinoma. EGFR and PSMA do show low staining levels in tumor tissue with urothelial carcinoma and do not seem suitable as target agents. Trial registration: The Medical Ethics Review Board of the University Medical Center Groningen approved this study on 14 December 2017 (METc UMCG 2017/639). Trial registration number (UMCG Research Register): 201700868.