Cyclin-dependent kinases (CDK) are key positive regulators of cell cycle progression and attractive targets in oncology. SCH 727965 inhibits CDK2, CDK5, CDK1, and CDK9 activity in vitro with IC₅₀ values of 1, 1, 3, and 4 nmol/L, respectively. SCH 727965 was selected as a clinical candidate using a functional screen in vivo that integrated both efficacy and safety parameters. Compared with flavopiridol, SCH 727965 exhibits superior activity with an improved therapeutic index. In cell-based assays, SCH 727965 completely suppressed retinoblastoma phosphorylation, which correlated with apoptosis onset and total inhibition of bromodeoxyuridine incorporation in >100 tumor cell lines of diverse origin and background. Moreover, short exposures to SCH 727965 were sufficient for long-lasting cellular effects. SCH 727965 induced regression of established solid tumors in a range of mouse models following intermittent scheduling of doses below the maximally tolerated level. This was associated with modulation of pharmacodynamic biomarkers in skin punch biopsies and rapidly reversible, mechanism-based effects on hematologic parameters. These results suggest that SCH 727965 is a potent and selective CDK inhibitor and a novel cytotoxic agent. Mol Cancer Ther; 9(8); 2344–53. ©2010 AACR.