Primary and metastatic human melanomas express muscarinic receptors. In embryonic tissues, expression of muscarinic receptors is correlated with morphogenesis. The hypothesis has been put forward that muscarinic receptors are involved in morphogenetic movements in the embryo, and in cellular movements in melanoma cells during invasive growth. The purpose of the present study was to characterize the muscarinic receptors in the human melanoma cell line SK-Mel 28 and to test in a Boyden chamber assay whether the chemotactic activity towards fibronectin can be influenced by muscarinic stimulation. In Western blots with the monoclonal antibody M35, muscarinic receptors were localized in a strong band at 66 kDa, and in a weak band at 63 kDa. Western blot with M3 subtype specific antibodies reproduced the line at 66 kDa. RT-PCR revealed mRNA for subtypes M3 and M5. These findings suggest that SK-Mel 28 cells express a large number of subtype M3 and a small number of subtype M5 receptors. Microscopic observation of calcium mobilization after muscarinic stimulation indicated that all cells carried functional muscarinic receptors. A standardized chemotaxis assay was established in modified Boyden chambers using fibronectin as chemotactic agent. After addition of carbachol to the upper compartment, an increase of fibronectin induced chemotaxis of ∼30% was observed, an effect abrogated by atropine. These results demonstrate that muscarinic cholinergic treatment has a modulatory effect on fibronectin-induced chemotaxis in SK-Mel 28 melanoma cells, indicating that the muscarinic system is involved in regulation of cell movement.