Detection of colorectal carcinomas with high-frequency microsatellite instability (MSI-H) is clinically important for several reasons. Recent studies suggested that immunohistochemical analysis of MLH1 and MSH2 expression is a rapid and accurate method for identifying large bowel tumors of the MSI-H phenotype. In this study, we evaluated by immunohistochemistry MLH1 and MSH2 protein expression in 132 MSI-H, 23 MSI-L (low-frequency MSI), and 150 microsatellite stable (MSS) colorectal adenocarcinomas. Loss of MLH1 or MSH2 expression was detected in 120 (90.9%) MSI-H carcinomas, whereas all MSI-L and MSS tumors showed normal expression of both proteins. Lack of MLH1 nuclear staining was observed much more often than absence of MSH2 nuclear staining (106 and 14 cases, respectively). Among MSI-H carcinomas, MLH1/MSH2 pattern of expression was significantly related to several clinical and pathological variables. In particular, MSI-H MLH1/MSH2-positive carcinomas were more often located in the distal colon, were more frequently classified as ordinary adenocarcinomas, and were more likely to be well or moderately differentiated, p53 positive, and <7 cm in diameter than were MLH1-negative and MSH2-negative carcinomas. In addition, MLH1-negative carcinomas were less common among patients with hereditary nonpolyposis colorectal cancer (HNPCC) or suspected HNPCC and in the group of patients aged <50 years. Patients with MLH1-negative carcinomas more frequently died of disease than did patients with MLH1/MSH2-positive and MSH2-negative MSI-H tumors, but the difference was not statistically significant. The results of the present investigation strongly indicate that immunohistochemical analysis of MLH1 and MSH2 expression is a practical and reliable method for the routine detection of the vast majority of MSI-H large bowel adenocarcinomas. Our data also point out that MSI-H MLH1/MSH2-positive colorectal carcinomas are characterized by distinctive pathological features.