To study invasion of lung cancer in vitro a novel three-dimensional coculture assay consisting of living human tissues has been developed. Multicellular spheroids initiated from a new large-cell lung carcinoma cell line (GaL23), found to be invasive in immunodeficient mice, were confronted with precultured bronchial fragments derived from mucosal biopsies obtained during routine fiberoptic bronchoscopy. The bronchial fragments consist of a stromal core with scattered fibroblasts covered by a continuous surface epithelium resting on a basal lamina. During the first 2 wk of confrontation, a gradual retraction of the bronchial epithelium with subsequent adhesion of the tumor cells to the underlying basal lamina occurred. The following week, a limited invasion of tumor cells into the bronchial stroma was seen. To facilitate the entrance of tumor cells through the mucosal surface, the surface epithelium was removed prior to coculture by ethylenediaminetetraacetic acid (EDTA) buffer treatment. Upon confrontation, GaL23 cells then rapidly attached to and migrated on the exposed basal lamina and an increasing number of tumor cells was seen in the stroma during the first week of culture. This model offers opportunities for studying mechanisms of lung cancer adhesion, migration, and invasion using human bronchial mucosa as the natural target tissue.