MUC1 is a transmembrane glycoprotein expressed on the apical surface of epithelial cells and exhibiting structural features characteristic of receptors for cytokines and growth factors. Its intracellular cytoplasmic tail (CT) contains multiple amino acid sequence motifs that, once phosphorylated, serve as docking sites for SH2 domain-containing proteins mediating signal transduction. Most studies examining MUC1 signaling have focused on cancer cells where MUC1 is overexpressed, aberrantly glycosylated, and constitutively phosphorylated. No studies have determined the signaling pathways activated in response to stimulation of its ectodomain. To better understand the signaling mechanisms of MUC1, we stably transfected HEK293 cells with an expression plasmid encoding a chimeric protein consisting of the extracellular and transmembrane domains of CD8 and the MUC1 CT (CD8/MUC1). Extracellular treatment of HEK293-CD8/MUC1 cells with CD8 antibody induced intracellular Tyr phosphorylation of the MUC1 CT and activated ERK1/2, but not the p38, SAPK/JNK, or ERK5 MAP kinases. Moreover, phosphorylation of ERK1/2 was completely blocked using a CT deletion mutant or a mutant construct in which all Tyr residues in the CT were changed to Phe. These results establish that Tyr phosphorylation of the MUC1 CT is required for activation of a downstream ERK1/2 pathway.