Formalin-fixed and paraffin-embedded tissues are a valuable resource for diagnosis and research. PCR is one of the most powerful methods of retrospective analysis of the DNA present in fixed tissues. One major problem with the molecular analysis of tissue samples, however, is cellular heterogeneity, ie, the large variety of cell types usually present in these specimens can mask cell-specific genetic alterations associated with disease. Herein we describe a procedure for obtaining and analyzing single cells recovered from stained histologic tissue sections without risking contamination from neighboring cells. An ultraviolet laser microbeam was used to physically destroy the tissue surrounding the single cells of interest. These cells, now freed from adjacent cells, were then easily retrieved with a motorized, computer-controlled micromanipulator and molecularly characterized through the use of PCR-based microanalysis. This accurate microdissection technique, followed by DNA amplification and direct sequencing, revealed a novel mutation in the gene coding for the cell adhesion molecule E-cadherin in single tumor cells that was absent in the adjacent single epithelial cells of a patient with early gastric cancer of the diffuse type. In this form of malignancy, tumor cells lose homophilic cell-to-cell interactions and invade the connective tissue as single cells. E-cadherin gene mutations have previously been detected in advanced diffuse-type gastric cancer and gastric carcinoma cell lines. The present study suggests that E-cadherin gene mutations may be an early event in gastric tumorigenesis. The laser-based isolation and subsequent molecular characterization of individual cells, as described herein, allows for micrometer-sized precision and should prove useful in detecting the nucleic acid abnormalities that underlie cancer, infection, and genetic disease.