Cytolytic T lymphocytes (CTL), natural killer cells, and lymphokine-activated killer (LAK) cells are cytolytic cells known to release the cytolytic protein perforin and a family of proteases, named granzymes, from cytoplasmic stores upon interaction with target cells. We now report the purification of an additional major 60-kD granule-associated protein (grp 60) from human LAK cells and from mouse cytolytic T cells. The NH2-terminal amino acid sequence of the polypeptide was found to be identical to calreticulin. Calreticulin is a calcium storage protein and carries a COOH-terminal KDEL sequence, known to act as a retention signal for proteins destined to the lumen of the endoplasmic reticulum. In CTLs, however, calreticulin colocalizes with the lytic perforin to the lysosome-like secretory granules, as confirmed by double label immunofluorescence confocal microscopy. Moreover, when the release of granule-associated proteins was triggered by stimulation of the T cell receptor complex, calreticulin was released along with granzymes A and D. Since perforin is activated and becomes lytic in the presence of calcium, we propose that the role of calreticulin is to prevent organelle autolysis due to the protein's calcium chelator capacity.