The precursor for IL-16 (pro-IL-16) is a nuclear and cytoplasmic PDZ domain-containing protein. In this study we have found that pro-IL-16 is absent or mutated in four T lymphoblastic leukemia cell lines examined. Ectopic expression of pro-IL-16 in pro-IL-16-negative Jurkat cells blocks cell cycle progression from G 0/G 1 to S phase associated with elevated levels of the cyclin-dependent kinase inhibitor p27 KIP1. Pro-IL-16 decreases p27 KIP1 degradation by reducing transcription and subsequent expression of Skp2, a key component of the SCF Skp2 ubiquitin E3 ligase complex. Taken together, these findings identify pro-IL-16 as a novel regulator of Skp2 expression and p27 KIP1 levels and implicate a role for pro-IL-16 in T cell proliferation.