In this report, we provide evidence that muscarinic receptors play a role in the generation of CD8+ cytolytic T lymphocytes. Analysis of mice with targeted deletions of each of the known muscarinic receptors (M 1-M5) showed that CD8+ T cells from M 1 receptor-deficient mice had a defect in the ability to differentiate into cytolytic T lymphocytes. Additional pharmacological experiments support the role of muscarinic receptors in wild type mice and suggest that acetylcholine may be involved. Together, these findings suggest that the M1 muscarinic receptor is involved in CTL development, thus providing novel insights into CD8+ T cell biology and the potential role of cholinergic signaling in immune regulation. © 2005 Elsevier B.V. All rights reserved.