In Jurkat T cells, the type 3 ryanodine receptor (RyR) was knocked-down by stable integration of plasmid expressing type 3 ryanodine receptor antisense RNA. Stable integration of the antisense plasmid in individual clones was demonstrated by PCR of genomic DNA, expression of antisense RNA by reverse transcriptase PCR, and efficiently reduced expression of type 3 ryanodine receptor protein by Western blot. Selected clones were successfully used to analyze T cell receptor/CD3 complex-mediated Ca(2+) signaling. Reduced expression of the type 3 RyR resulted in (i) significantly decreased Ca(2+) signaling in the sustained phase and (ii) in permeabilized cells in a significantly impaired response toward cyclic ADP-ribose but not to d-myo-inositol 1,4,5-trisphosphate. For the first time, the role of the type 3 RyR in sustained Ca(2+) signaling was directly visualized by confocal Ca(2+) imaging as a significant contribution to the number and the magnitude of subcellular Ca(2+) signals. These data suggest that the type 3 ryanodine receptor is essential in the sustained Ca(2+) response in T cells.