Molecular analysis of microdissected tissue samples is used for analyzing tissue heterogeneity of histological specimens. We have developed a rapid one-step microdissection technique, which was applied for the selective procurement of tissue areas down to a minimum of 10 cell profiles. The special features of our microdissection system consist of an ultrasonically oscillating needle and a piezo-driven micropipette. The validity of this technique is demonstrated in human lung large-cell carcinoma by real-time quantitative reverse transcriptase-polymerase chain reaction assays of vimentin, cyclin D1, and carcinoembryonic antigen after linear RNA amplification. mRNA expression values of microdissected samples scattered around those of bulk tumor tissue and showed differential mRNA expression between samples of tumor parenchyma and supportive stromal cells for vimentin and carcinoembryonic antigen as confirmed by immunohistochemistry. In conclusion, this procedure requires simple equipment, is easily performed, and delivers microdissected tissue samples of oligocellular clusters suitable for further molecular analysis.