It is becoming increasingly clear that no single marker will have the sensitivity and specificity necessary to be used on its own for diagnosis/prognosis of tumors. Inter-patient and intra-tumor heterogeneity provide overwhelming odds against the existence of such an ideal marker. With this in mind our laboratory has been applying a long-term systematic approach to identify multiple biomarkers that can be used for clinical purposes. As a result of these studies, we have identified and reported several candidate biomarker proteins that are deregulated in bladder cancer. Following the conceptual biomarker development phases proposed by the Early Detection Research Network we have taken some of the most promising candidate proteins into post-discovery validation studies and here we report on the characterization of one such biomarker, the Bladder Cancer Associated Protein (BLCAP), formerly termed Bc10. In order to characterize BLCAP protein expression and cellular localization patterns in benign bladder urothelium and UCs we used two independent sets of samples from different patient cohorts; a reference set consisting of 120 bladder specimens (formalin-fixed as well as frozen biopsies), and a validation set consisting of 2108 retrospectively collected UCs with long-term clinical followup. We could categorize the UCs examined into four groups based on levels of expression and sub-cellular localization of BLCAP protein, and showed that loss of BLCAP expression is associated with tumor progression. The results indicated that increased expression of this protein confers an adverse patient outcome, suggesting that categorization of staining patterns for this protein may have prognostic value. Finally, we applied a combinatorial two-marker discriminator using BLCAP and A-FABP, another UC biomarker previously reported by us, and found that the combination of the two markers correlated more closely with grade and/or stage of disease than the individual markers. The implications of these results in biomarker discovery are discussed.