The matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) have been associated with tumor invasion and metastasis in many human cancers. Immunohistochemical studies were performed on frozen tumor samples from 42 patients with invasive bladder cancer treated by cystectomy with monoclonal antibodies against the Mr 72,000 gelatinase A (MMP-2), Mr 92,000 gelatinase B (MMP-9), and TIMP-2 to evaluate their significance in bladder cancer. Immunoreactivity for the gelatinases was predominantly tumor cell-associated, whereas strong TIMP-2 staining was mostly detected in the stroma. Tumor cells demonstrated moderate to strong reactivity for MMP-2 and MMP-9 in 71 and 71% of cases, respectively, which did not correlate with stage, grade, or outcome. Tumor cells were positive for TIMP-2 in 26 (62%) of 42 cases, and this correlated with a worse outcome (69 versus 25% died of disease; P < 0.05). In 31 (74%) of 42, there was moderate to strong stromal staining for TIMP-2; this also was associated with a poor outcome (65 versus 25% died of cancer; P < 0.05). Tumor basement membrane (BM) status was investigated using an antibody to type IV collagen. In 9 cases, the invasive tumor nests were surrounded by an intact BM; in 7 of these, stromal staining for TIMP-2 was absent. None of these 9 patients (0%) died of tumors compared with 7 (100%) of 7 with complete loss of BM staining (P < 0.001). These results suggest a potential role for TIMP-2 and BM staining as prognostic indicators in invasive bladder cancer.