We have recently demonstrated that killer cells expressing a cytolytic factor, perforin, infiltrate the hearts of mice with acute viral myocarditis and may play an important role in the mechanism of myocardial damage. To clarify the mechanism of in vivo cardiac myocyte injury mediated by perforin, we investigated the release of perforin molecules from killer cells by immunoelectron microscopy and examined the circular lesions formed by perforin on the membrane of cardiac myocytes. We found that there was massive release of perforin molecules from the killer cells directly onto the surface of the cardiac myocytes. Furthermore, electron microscopy of ultrathin ventricular sections treated with trypsin revealed numerous circular lesions with the characteristics of perforin pores, in the membranes of cardiac myocytes. These findings provide the first direct evidence that killer cells injure cardiac myocytes by releasing perforin and may play a critical role in the myocardial damage occurring in acute viral myocarditis.