We demonstrated that eosinophilic leukemia cell line-1 (EoL-1 cells) differentiated into monocytic/macrophagic cells by a treatment with interferon-gamma. Muscarinic receptor mRNA was not detected in untreated EoL-1 cells, and the cytosolic concentration of Ca2+ ([Ca2+]i) did not rise either in these cells. Interestingly, when EoL-1 cells were treated with interferon-gamma, mRNAs for muscarinic M3 and M5 receptors could be detected in these cells, along with an increase in [Ca2+]i and chemotaxis induced by carbachol that could be blocked with 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and pirenzepine. These data support the functional importance of muscarinic M3 and M5 receptors in monocytic/macrophagic cells differentiated from EoL-1 cells. This model also provides evidence of a significant functional interaction between muscarinic M3 and M5 receptors.