Progressive loss of salivary gland function occurs in most patients undergoing head and neck radiotherapy. It is unclear whether adult salivary gland tissue contains stem/progenitor cells. In this study, we used a colony assay to clarify the presence of stem/progenitor cells in adult submandibular glands after irradiation. We developed a novel culture system that promotes single-cell colony formation with low density culture of irradiated and non-irradiated adult human submandibular gland cells using serum-free medium following serum-supplemented medium. The cells from all samples, except those obtained from the oldest patient who received the highest radiation dose, expressed acinar, ductal, and myoepithelial cell-lineage markers with reverse transcription-polymerase chain reaction (RT-PCR) and immunostaining. A sub-culture of these colonies with serum-free medium showed high multipotency. These results are the first description of presence of salivary gland stem/progenitor cells with self-renewal, high proliferation and multipotent differentiation activity in salivary glands, even after irradiation. The survival of the cells depends on radiation dose and cell aging.