Interleukin-16, a proinflammatory cytokine produced in CD8⁺ lymphocytes, is synthesized as a precursor protein (pro-IL-16). It is postulated that the C-terminal region of pro-IL-16 is cleaved, releasing bioactive IL-16. To characterize IL-16 cleavage, we transfected COS cells with a cDNA encoding a ∼50-kDa form of pro-IL-16. Transfected COS cells released a ∼20-kDa IL-16 cleavage product shown to consist of the 121 C-terminal residues of pro-IL-16 by immunoblotting and amino acid sequencing. Cleaved IL-16, but not pro-IL-16, exhibited lymphocyte chemoattractant activity. A C-terminal ∼20-kDa IL-16 polypeptide was also released when pro-IL-16 was treated with concanavalin A-stimulated CD8⁺ lymphocyte lysate. Cleavage occurred after an Asp, suggesting involvement of a caspase (interleukin-1β-converting enzyme/CED-3) family protease. Using recombinant caspases and granzyme B, we determined that pro-IL-16 cleavage is mediated only by caspase-3. Relevance to pro-IL-16 processing in primary lymphocytes was supported by identifying the p20 subunit of activated caspase-3 in stimulated CD8⁺ lymphocytes and by inhibition of CD8⁺lymphocyte lysate-mediated cleavage with Ac-DEVD-CHO. Pro-IL-16 is a substrate for caspase-3, and cleavage by this enzyme releases biologically active IL-16 from its inactive precursor.