Microsatellite instability (MSI) is characteristic of hereditary nonpolyposis colorectal cancer (HNPCC). Owing to early onset of colorectal cancer before the age of 50 years and/or familial clustering of HNPCC-related malignancies, the diagnosis of HNPCC was suspected in 2 patients. Because no paraffin-embedded tumor tissue was available, we used archival 5-microm, hematoxylin-eosin-stained tumor specimen slides for direct MSI analysis. Tissue was microdissected and cells were lysed using 1% Triton. Fluorescence polymerase chain reaction amplification of a panel of 7 microsatellite markers, including all markers of the current international reference panel (BAT-25, BAT-26, D2S123, D5S346, and D17S250), demonstrated MSI in one patient and excluded MSI in the other. In conclusion, this study demonstrates the feasibility of MSI analysis by direct fluorescence polymerase chain reaction amplification using hematoxylin-eosin-stained tissue specimens without the need for prior DNA extraction.