The development of ovarian cancer, unlike that of most human tumors, is rarely dependent upon the mutually exclusive loss of RB and p16 cell cycle proteins. RB+/p16+ ovarian cancer cell lines are, however, insensitive to the growth-suppressive effects of ectopically expressed p16 protein, which suggests that they harbor as yet unidentified defects that compromise cell cycle regulation in late G1/S. In the current study, we used Western blotting to analyze cyclin E protein expression in a panel of normal and tumor ovarian tissues and ovarian cancer cell lines (including the p16-insensitive RB+/p16+ ovarian cancer cell line, NIH-OVCAR-3). Both the NIH-OVCAR-3 cell line and 70% of RB+/p16+ ovarian tumors showed abnormally elevated levels of the full-length cyclin E protein (EL1) in addition to several low molecular weight (LMW) isoforms of cyclin E. Using small interference RNA (siRNA), we have inhibited the synthesis of cyclin EL1 protein by approximately 80% and eliminated the LMW isoforms in NIH-OVCAR-3 ovarian cancer cells. Associated with the down-regulation of cyclin E expression, we observed both a marked shift in RB protein expression to the active, hypophosphorylated state and barely detectable expression of cyclin A (which is usually expressed upon entry into S-phase). Consistent with the protein expression data, cell cycle distribution analysis indicated that the NIH-OVCAR-3 cells had undergone a marked accumulation in G1 phase of the cell cycle. These data indicate the therapeutic potential of targeted RNA interference in the treatment of ovarian cancer patients whose tumors overexpress cyclin E protein.