Prointerleukin-16 (Pro-IL-16) is an abundant, PDZ domain-containing protein expressed in the nucleus and cytoplasm of resting human T lymphocytes. We have previously shown that ectopic expression of Pro-IL-16 in Pro-IL-16-negative human Jurkat cells represses transcription of the F-box protein, Skp2, resulting in accumulation of the cyclin-dependent kinase inhibitor, p27(Kip1), and G0/G1 cell cycle arrest. The current studies demonstrate the kinetics of Pro-IL-16 and p27(Kip1) expression in activated normal human T lymphocytes. We correlate nuclear Pro-IL-16 loss with decreased p27(Kip1) expression, increased cell cycle progression, and proliferation. Conversely, we show that constitutive expression of Pro-IL-16 by retroviral infection of activated human T lymphocytes induces G0/G1 cell cycle arrest, inhibits proliferation, and is associated with increased levels of p27(Kip1). These findings implicate nuclear Pro-IL-16 as a cell cycle regulatory protein for human T lymphocytes.