For unknown reasons, the pilosebaceous unit displays prominent alkaline phosphatase (AP) activity, and alterations in AP activity are seen in alopecia areata. The role of AP in hair biology and pathology has been obscured by contradictory reports on the localization and activity of AP during the hair cycle, and by a paucity of instructive models for studying AP functions. Using the C57 BL-6 mouse model for hair research, we have characterized endogenous AP with a simple histochemical developing solution routinely employed for AP immunohistology. This method was selective for AP, and revealed distinctive hair cycle-dependent changes in AP activity and localization. Although the dermal papilla displays unusually strong AP activity during the entire hair cycle, the outer root sheath is AP-positive only during late anagen and early catagen. Strong, rather homogeneous AP activity is seen in the sebaceous gland (SG) only during catagen and telogen. This AP staining pattern indicates hair cycle-dependent changes in SG functions, and differs to some extent from the previously reported AP activity during the hair cycle of various species. We propose a simple and effective technique for follicle classification based on the AP histochemistry of dermal papilla and sebaceous gland, and discuss uses of the C57 BL-6 mouse model for functional AP studies.