Carcinoma-stromal cell interaction and air-liquid interface (ALI) constitute a specific microenvironment that modulates the biological behavior of carcinoma cells of the upper aerodigestive tract. Although radiotherapy is a useful tool for treating carcinomas of this organ, effects of irradiation on carcinoma cells under carcinoma-stromal cell interaction and ALI are unclear. To address this issue, we examined the effects of irradiation on the proliferation and apoptosis of squamous cell carcinoma cell lines (KB and HEp-2), using three-dimensional collagen gel culture with both carcinoma-stromal cell interaction and ALI. During the second week after irradiation with or without the two factors mentioned above, bromodeoxyuridin (BrdU) uptake and apoptosis of KB, and HEp-2 cell types decreased and increased, respectively. After this stage, the carcinoma cells with these two factors actively re-proliferated together with increased BrdU uptake and decreased apoptosis, whereas the magnitude of these parameters was considerably lower in culture without these factors. We applied our method to carcinoma tissues obtained from several clinical cases. At the same stage, the irradiated carcinoma cells replicated the phenomena observed in cell lines. The data indicate that carcinoma-stromal cell interaction and ALI together promote the re-proliferation of irradiated carcinoma cells and their decreased apoptosis, suggesting that our method is a possible model for testing radiosensitivity of carcinomas in a more physiological condition.