Objective: Enterovirus infections are major etiological factors in myocarditis and dilated cardiomyopathy. Using an experimental murine model of this disease, previous studies have shown that myocarditis susceptibility depends upon activation of T lymphocytes expressing the γδ T cell receptor (TcR), and that only mouse strains which accumulate γδ T cells in the myocardium show apoptosis of myocytes or evidence of dilated cardiomyopathy-like disease. The objective of the present studies is to demonstrate that γδ T cells directly induce greater Fas-dependent apoptosis of cultured myocytes than T cells expressing the αβ TcR. Methods: Bl.Tg.Eα mice were infected for 7 days with coxsackievirus B3 (CVB3). Hearts were removed and were either formalin-fixed, sectioned and stained with hematoxylin and eosin for inflammation, and using TdT-TUNEL for apoptosis, or were minced and collagenase digested for isolation of γδ+ and αβ+ T cells using immunomagnetic bead separation. Neonatal cultures of cardiac myocytes were isolated from mice less than 2 days old by collagenase and pancreatin digestion, and were either untreated or infected with virus. Levels of Fas (CD95) were measured using FITC-conjugated hamster anti-mouse Fas monoclonal antibody and flow cytometry. Susceptibility of myocytes to Fas-dependent killing was measured by ⁵¹Cr-release by labeled myocytes incubated for 4 h on either 3T3-mock or 3T3-FasL transfected cell monolayers. Killing by T cells was also measured in a 4 h ⁵¹Cr-release assay. Fas-dependent and perforin-dependent cytotoxicity was determined by specific blocking using either Fas-Fc or concanamycin A. Results: Virally infected myocyte cultures showed significantly enhanced Fas expression compared to uninfected cells, with maximal upregulation of Fas occurring 18–24 h after virus infection. Both infected and uninfected myocytes were selectively killed by FasL-transfected 3T3 cells but not by mock control cells. Approximately 38% of CD3+ lymphocytes isolated from the heart express the γδ TcR with the remainder expressing the αβ TcR. Both γδ+ and αβ+ T cells lysed myocyte targets. Blocking studies indicate that γδ+ T cells induced predominantly Fas-mediated killing, while αβ+ cell produced more perforin-mediated death, although these effectors were capable of Fas-dependent killing as well. Conclusions: These studies demonstrate that T cells expressing the γδ TcR are more effective mediators of myocyte apoptosis than αβ+ T cells in vitro and suggests that these effectors may be primarily responsible for myocardial injury associated with dilated cardiomyopathy-like signs during coxsackievirus B3-induced myocarditis.