Although widely studied as a neurotransmitter, Tcell–derived acetylcholine (ACh) has recently been reported to play an important role in regulating immunity. However, the role of lymphocyte-derived ACh in viral infection is unknown. Here, we show that the enzyme choline acetyltransferase (ChAT), which catalyzes the rate-limiting step of ACh production, is robustly induced in both CD4+ and CD8+ Tcells during lymphocytic choriomeningitis virus (LCMV) infection in an IL-21–dependent manner. Deletion of Chat within the Tcell compartment in mice ablated vasodilation in response to infection, impaired the migration of antiviral Tcells into infected tissues, and ultimately compromised the control of chronic LCMV clone 13 infection. Our results reveal a genetic proof of function for ChAT in Tcells during viral infection and identify a pathway of Tcell migration that sustains antiviral immunity. [ABSTRACT FROM AUTHOR]