A3 was generated as an antibody recognizing somatic stem cells in rat tissues. We investigated the distribution of A3-positive cells in developing rat hair follicles by immunolabeling. A3-positive cells began to be seen in the hair germ and peg in fetuses and neonates; the positive cells were epithelial cells above basal cells. Furthermore, A3-positive cells were seen in the outer root sheath adjacent to the bulge in mature hair follicles. Double immunofluorescence revealed that these A3-positive epithelial cells reacted to E-cadherin (for all epithelial elements) but not to CK15 (for basal cells/epithelial stem cells) or to nestin (for stem cells), indicating that A3-positive epithelial cells are suprabasal cells in the developing epidermic hair follicle. Additionally, spindle-shaped mesenchymal cells surrounding the hair peg and mature hair follicle reacted to A3; in double immunofluorescence, the A3-positive cells were located outside collagen type IV-positive glassy membrane, and reacted to vimentin (for mesenchmal cells), Thy-1 (for immature mesenchymal cells), CD34 (for stem cells) and nestin, but not to α-smooth muscle actin (for myofibroblasts); the positive cells were regarded as immature mesenchymal cells with stem cell nature in the connective tissue sheath of developing hair follicles. A3-positive epithelial and mesenchymal cells did not show proliferating activity. Collectively, it is considered that A3-positive cells seen in developing rat hair follicles may be quiescent post-progenitor cells with the potential to differentiate into either highly-differentiated epithelial or mesenchymal cells. A3 would become a useful antibody to know the kinetics of rat hair follicle-constituting cells.