—Cytotoxic T lymphocytes (CTLs) that infiltrate the heart are important immune effectors implicated in heart transplant rejection, myocarditis, and other cardiomyopathies. To investigate the mechanism(s) underlying CTL damage to the myocardium through activation of the Fas receptor (Fas/CD95/Apo-1) by the Fas ligand, we explored the interaction between peritoneal exudate CTLs (PELs), derived from perforin gene–knockout (P−/−) mice, and murine ventricular myocytes. Fas expression on isolated ventricular myocytes was demonstrated immunohistochemically. Action potentials, [Ca²⁺]_i transients, and contractions of myocytes conjugated to P−/− PELs or treated with the apoptosis-inducing anti-Fas monoclonal antibody Jo2 were recorded. Action potential characteristics of nonconjugated myocytes and myocytes conjugated with P−/− PELs were, respectively, as follows: V_m, −73.2±1.5 and −53.6±6.4 mV (mean±SEM); action potential amplitude, 117.9±3.9 and 74.3±21.2 mV; and action potential duration at 80% repolarization, 17±6 and 42±13 milliseconds (all P<.05). P−/− PELs also induced early and delayed afterdepolarizations as well as arrhythmogenic activity. Diastolic [Ca²⁺]_i increased during the cytocidal interaction with P−/− PELs, from a fluorescence ratio of 0.82±0.05 (n=7) to 1.98±0.09 (n=13) (P<.05). All of the effects caused by P−/− PELs were reproduced by incubating the myocytes with Jo2. Heparin (50 μg/mL), an antagonist of inositol trisphosphate (IP₃)–operated sarcoplasmic reticulum Ca²⁺ channels, or U-73122 (2 μmol/L), a phospholipase C inhibitor, but not the inactive agonist U-73343, prevented Fas-mediated myocyte dysfunction. Additionally, intracellular application (through the patch pipette) of the active IP₃ analogue, inositol 1,4,5-trisphosphate, but not the inactive analogue, inositol 1,3,4-trisphosphate, caused electrophysiological changes resembling those resulting from P−/− PELs and Jo2, suggesting that CTL-induced Fas-based myocyte dysfunction is mediated by IP₃. We conclude that a Fas-based perforin-independent mechanism of CTL action can account for the immunopathology seen in the allotransplanted heart, myocarditis, and dilated cardiomyopathy.