Three-dimensional organ cultures allow performing research in vitro in a complex multi-cellular environment. We aimed at developing a long term coculture system (COC) for the study of lung cancer to repeatedly measure tumor volume. Organ cultures of bronchial mucosa with 1-2 mm diameter were embedded in agarose and bisected with a tissue slicer so that the organ culture within was cut into halves uncovering the connective tissue of the stroma of each half. A cell suspension of GFP-transfected EPLC 32M1 lung tumor cells was brought in contact with the connective tissue of the wounded surface. Adherent tumor cells grew invasively into the organ culture. Using 2-Photon microscopy, Z-stacks were recorded, reconstructed with appropriate analysis software, and the tumor volume was calcvulated. Tumor cells were identified by GFP-fluorescence. Repeated measurements of the same COC could be performed over up to 8 weeks. The tumor volume increased continually with the growth rate becoming slower towards the end of culture. A comparison of two clones of tumor cells which had shown different rates of proliferation in monolayer culture demonstrated that the clone with the higher rate of proliferation in monoculture produced tumors with more rapid growth in the COC model. In this study we present a coculture system for the study of lung cancer using 2-Photon microscopy. COCs are particularly appropriate for long term in vitro treatment studies.