Cholinergic receptors of the muscarinic class (M1-M5) are expressed in epidermal keratinocytes and melanocytes as well as in the hair follicle. Knockout (KO) mice of all five receptors have been created and resulted in different phenotypes. KO mice with a deletion of the M4 muscarinic acetylcholine receptor (M4R) present a striking hair phenotype, which we have analyzed here in greater detail by quantitative histomorphometry. Earlier studies revealed a retarded hair follicle morphogenesis in M4R KO mice, compared to age-matched wild type controls. On day 17, when mice enter the first hair growth cycle, the KO mice still showed a slightly retarded catagen phase. Subsequently, hair follicles of the KO mice stayed in a highly significantly prolonged telogen phase, while wild type mice had already far progressed in the hair cycle by entry into anagen. Most strikingly, the M4R KO mice did not engage in follicular melanogenesis and failed to produce pigmented hair shafts. The current pilot study suggests that the M4R plays a fundamental role in the control of the murine hair follicle cycling and is an essential signaling element in the control of hair follicle pigmentation.