Side population (SP) cells are characterized by their ability to efflux the vital dye Hoechst 33342 due to expression of the ATP binding cassette (ABC)-dependent transporter ABCG2, and are highly enriched for stem/progenitor cell activity. In this study, we identified SP cells in murine thyroid, which are composed of two populations of cells; CD45(-)/c-kit(-)/Sca1(+) and CD45(-)/c-kit(-)/Sca1(-) cells. Quantitative RT-PCR analysis revealed that SP cells highly express ABCG2 and the stem cell marker genes encoding nucleostemin and Oct4, while the expression of genes encoding the thyroid differentiation markers, thyroid peroxidase, thyroglobulin, and thyrotropin receptor, and two transcription factors, Titf1 and Pax8, critical for thyroid specific gene expression, are low in SP cells as compared with the main population (MP) cells. In situ hybridization and double immunofluorescence demonstrated that cells expressing Abcg2 gene reside in the interfollicular space of the thyroid gland. Approximately one-half and a small percentage of the ABCG2-positive cells were also positive for vimentin and calcitonin, respectively. After 9 weeks under three dimensional thyroid primary culture conditions, MP cells formed an epithelial arrangement and follicle-like structures that are immunoreactive for TITF1 and thyroglobulin. In contrast, SP cells demonstrated very few morphological changes without any epithelial or follicle-like structure and negative immunostaining for TITF1 and thyroglobulin. These results demonstrate that thyroid possesses SP cells that may represent stem/progenitor cells.