Progression of the eukaryotic cell division cycle is regulated by a series of structurally related serine/threonine protein kinases known as cyclin-dependent kinases (CDKs). The D-type cyclin-dependent kinases, CDK4 and CDK6, have been strongly implicated in the control of G1 progression and the phosphorylation of the retinoblastoma protein, pRb. The formation of complexes and enzymatic activity of cyclin D-CDK4 and cyclin D-CDK6 kinases is negatively regulated by p16INK4 (MTS1/CDK4I/CDKN2) via its specific interaction with CDK4 and CDK6 catalytic subunits. Here we report that the p16 mRNA accumulates to a high level in cells lacking pRb function and transcription of p16 is repressed by pRb. Our results provide evidence supporting a feedback regulatory loop involving pRb, p16, and cyclin-dependent kinases.