We have identified previously six nuclear matrix proteins (NMPs) that are bladder cancer specific. In this study, we analyzed the expression of one of these proteins, BLCA-4, in bladder tumors and normal bladder tissue. We also examined the appearance of BLCA-4 in the urine as a biomarker for bladder cancer. BLCA-4 was isolated from nuclear matrix preparations of bladder tumors, and its peptide sequence was determined. The antibodies generated against the resulting BLCA-4 peptides were then used to detect its presence in immunoblots and in urine samples by immunoassay. We analyzed tissue samples of bladder tumor and normal donor bladders and urine obtained from 51 normal individuals and 54 patients with pathologically confirmed bladder cancer. The BLCA-4 peptide sequences do not resemble any known human protein sequences. On immunoblot analysis, BLCA-4 expression was detectable in tumor and normal tissues from patients with bladder cancer but not in any of the normal bladder tissue obtained from organ donors. Using a prospectively determined cutoff level of 13 A (absorbance) units/microg protein, all 51 normal individuals tested were negative for BLCA-4 expression, whereas 53 of 55 samples from patients with bladder cancer were positive. These results suggest that BLCA-4 is present throughout the bladder in both the tumor and morphologically normal areas in bladder cancer patients. BLCA-4 is a very sensitive (96.4%) and specific (100%) marker for bladder cancer. BLCA-4 is a bladder cancer-specific marker that can be detected using a urine-based assay and can be used in the diagnosis of bladder cancer.