Antigen-specific T cells demonstrate several potent effector functions during immune responses. Direct killing of infected cells is crucial for clearing viruses and other intracellular pathogens, but it has been difficult to measure the frequency of cytolytic cells. We have now developed a single-cell assay to measure the number of cytotoxic cells in a population, using a herpes simplex virus amplicon vector to express Escherichia coli β-galactosidase in mouse or human target cells, and an Elispot to detect release of β-galactosidase from killed target cells. This antigen-specific, perforin-dependent Lysispot assay has been combined with a cytokine Elispot in a two-color assay to confirm that cytotoxicity and interferon-γ secretion are regulated independently. The simultaneous enumeration of cytokine-secreting and cytotoxic cells should be invaluable for ex vivo analysis of immune responses during infection and autoimmunity.