The short-term kinetics of the interaction between mouse cytolytic T lymphocytes (CTL) and 51Cr-labeled target cells was investigated. It was found that addition of EDTA to mixtures of CTL and target cells instantaneously blocked de novo lytic interactions, but did not inhibit the release of 51Cr from already damaged target cells. Using this information, a modified cytolytic assay was developed. By applying this assay to highly active CTL populations generated in secondary mixed leukocyte cultures, it was possible to detect appreciable target cell damage as early as 30 seconds after exposure to CTL. Quantitative studies demonstrated linear relationships between cytolysis and time and between rate of cytolysis and cell number under these assay conditions.