Objective To study the self-renewal potential of Nestin-immunoreactive cells in mouse anterior pituitary. Methods Primary Nestin-immunoreactive cells were isolated from mouse anterior pituitary and enriched in specific culture condition. In order to analyze colony forming potential of Nestin-immunoreactive cells，secondary generation Nestin-immunoreactive mouse anterior pituitary cells were cultured in colony formation medium. For further confirming single cell origin of new colonies，cells isolated from both wild type and EGFP mice were mixed in culture condition as to trace the source of newly generated colonies. In addition，BrdU incorporation assay was utilized to verify that colonies were formed by cell proliferation，and then to evaluate proliferative rate of cells in colonies. Results Cells derived from primary culture condition with Nestin-immunoreactive cell morphology were detected to express nestin by immuncytochemistry. In conoly culture condition with very low cell density，1% of secondary generation Nestin-immunoreactive cells were capable of forming colonies EM>in vitro/EM>. Mixture culture of EGFP and wild type cells showed that colony derivatives under this condition were either homogenous EGFP cells or homogenous wild type cells，wherease there was no mixed colony detected. Moreover，BrdU incorporation assay demonstrated that within 24 hours 13% cells from colonies were generated from cell division (EM>n/EM>=20). Conclusion Mouse anterior pituitary contains Nestin-immunoreative cells. These cells are