Confocal laser scanning microscopy (CLSM) has become an exciting new instrument with rapidly expanding potential for application to the morphological examination. As an initial step of examining the possible values or potentials of CLSM observations in diagnostic pathology materials, we applied CLSM to the analysis of immunolocalization of proliferating cell nuclear antigen (PCNA), p53 and cytokeratin, and eosin and DNA fluorochrome propidium (PI) stain in cell smears obtained from 20 cases of squamous cell carcinoma of the esophagus. Superior contrasts and resolution were obtained in confocal images than in nonconfocal ones in immunocytochemistry, eosin, and PI stain. In immunocytochemistry, CLSM demonstrated subcellular localization of antigens examined, cytokeratin as coarse and fine intracytoplasmic fibers, PCNA as diffuse intranuclear localization, and p53 as heterogeneous intranuclear localization which appeared to be associated with chromatin structure. Optical sectioning of a specimen by the rejection of out-of-focus noise revealed three dimensional structure of cell clusters of squamous cell carcinoma. With eosin and PI as dyes for stain, three dimensional structures of any clusters on cell smears can be obtained. CLSM has vast potentials in the analysis of diagnostic cytology materials, including immunocytochemistry.