Cellular immunity plays a major role in the control of human cytomegalovirus (HCMV) infection. CD4(+) T lymphocytes have been shown to contribute to this function but their precise role is a matter of debate. Although CD4(+) T cells have been shown to kill target cells through the perforin/granzyme pathway, whether HCMV-specific CD4(+) T cells are capable of killing HCMV-infected targets has not yet been documented. In the present paper, we have taken advantage of well established cellular reagents to address this issue. Human CD4(+) T-cell clones specific for the major immediate-early protein IE1 were shown to perform perforin-based cytotoxicity against peptide-pulsed targets. However, when tested on infected anitgen presenting cell targets, cytotoxicity was not detectable, although gamma interferon (IFN-gamma) production was significant. Furthermore, cytotoxicity against peptide-pulsed targets was inhibited by HCMV infection, whereas IFN-gamma production was not modified, suggesting that antigen processing was not altered. Remarkably, degranulation of CD4(+) T cells in the presence of infected targets was significant. Together, our data suggest that impaired cytotoxicity is not due to failure to recognize infected targets but rather to a mechanism specifically related to cytotoxicity.