Acetylcholine and its receptors are not only essential for the nervous system but also act as mediators of cell communication between non-neuronal cells. Signal transduction after cholinergic stimulation is mediated by two types of receptors. The nicotinic acetylcholine receptors are ion channels, whereas the muscarinic receptors belong to the group of G protein coupled receptors (GPCR). Their activation can lead to initiation of the mitogen-activated protein (MAP) kinase cascade which contributes to cell survival, differentiation and other important cellular responses. The non-neuronal acetylcholine plays a substantial role in the human skin and regulates cell adhesion and migration, as well as proliferation and differentiation of keratinocytes. We here show that in the human keratinocyte cell line HaCaT, the muscarinic acetylcholine receptors are mediators of mitogenic signaling. Stimulation with the cholinergic agonist carbachol leads to an extensive activation of the MAP kinase ERK, together with an activation of the protein kinase Akt. These signaling pathways are dependent on the transactivation of the epidermal growth factor receptor (EGFR), and EGFR transactivation even appears to be the only pathway through which muscarinic receptors facilitate ERK activation in these cells. The transactivation pathway involves a triple-membrane-passing process, proceeding through the activation of matrix metalloproteases and an extracellular EGF-like ligand release.