Gene expression profiling with nucleic acid arrays is a powerful method for the molecular classification of human neoplasms. Laser capture microdissection is an equally useful technique to isolate defined cell populations selectively from heterogeneous histological tissue sections. We demonstrate how a modest use of laser capture microdissection is sufficient to isolate nanogram quantities of high-quality RNA. Together with several internal standards and microcapillary electrophoresis of input RNA, we used two rounds of linear molecular amplification to generate sufficient quantities of labeled target for hybridization to high-density oligonucleotide expression arrays. Our results demonstrate that the technique is reproducible, generates only modest biasing of the original transcript population and offers sensitivity comparable to that achieved using standard methodology. Using this approach, we have compared the expression profiles of nonmalignant human breast epithelium and adjacent ductal carcinoma in situ lesions from breast cancer patients. Several genes, including those previously implicated in human breast cancer progression, demonstrate differential expression among the microdissected cell populations. This study demonstrates the feasibility of applying laser capture microdissection and nucleic acid expression array technology to histologically complex clinical specimens to classify human breast cancer molecularly and gain a better understanding of its molecular pathobiology.