Cloned cytotoxic T lymphocytes (CTL) can lyse target cells in a Ca2+-dependent or Ca2+-independent fashion, depending on the target cell used. We have used these Ca2+-free assay conditions to determine whether there is serine esterase release in the absence of extracellular Ca2+. We find that under conditions where there is significant target cell killing in the absence of Ca2+, there is no detectable serine esterase release. To the extent that serine esterase and perforin-like molecules reside in the same granules, these results also suggest that target cell killing may occur in the absence of degranulation. Our results provide an example of target cell killing without serine esterase release, and the first indication that lysis and degranulation can be functionally dissociated.