Activation through TCR/CD3-plus-CD28 induces primary T lymphocytes to enter S phase. Downregulation of cyclin-dependent kinase inhibitor p27(kip1) is critical in this process and is mediated by ubiquitin-targeted degradation of p27(kip1). Ubiquitination of p27(kip1) is performed by the SCF(skp2) ubiquitin ligase comprised of the core components Roc1, Cul1 and Skp1 and the substrate recognition components Skp2 and Cks1. Here we show that in primary human T lymphocytes, the SCF(skp2) core components Roc1, Cul1 and Skp1 are constitutively expressed, and their levels remain unchanged upon TCR/CD3-plus-CD28 costimulation. In contrast, the substrate recognition components Skp2 and Cks1 are almost undetectable in resting T cells and are transcriptionally induced upon costimulation. We determined that the SKP2 promoter lies directly upstream of the translational start site and contains binding sites for SP1, Elk-1 and E2F transcription factors. Mutagenesis of SP1 and Elk-1 sites abrogated TCR/CD3-plus-CD28-mediated SKP2 promoter-driven reporter activity, whereas mutagenesis of an E2F site enhanced reporter activity, suggesting that SKP2 promoter may act as a node of integration for mitogenic and anti-mitogenic signals. Thus, in primary T lymphocytes CD28 costimulation can directly regulate cell cycle progression by inducing transcription of the substrate recognition components of SCF(skp2) ubiquitin ligase that targets p27(kip1) for degradation.