Background: Expression of polo-like kinase-1 (PLK1), which has several functions in mitotic progression, is elevated in a broad range of human tumors. To investigate the role of PLK1 in neoplastic proliferation, we used the technique of RNA interference. Methods: Cells from several different cancer cell lines (MCF-7 breast cancer cells, HeLa S3 cervical cancer cells, SW-480 colon cancer cells, and A549 lung cancer cells) were transfected with small interfering (si) RNAs targeted against the human PLK1 or lamin genes. Northern and western blot analyses were used to examine PLK1 gene expression in transfected cancer cells and normal cells (human mammary epithelial cells [HMECs]). The phenotype, proliferation, and cell cycle distribution of cells transfected with siRNAs were also monitored by fluorescence microscopy and fluorescence-activated cell sorting analysis. Results: All cancer cell lines transfected with low doses of siRNAs targeted to PLK1 had greatly decreased levels of PLK1 mRNA and protein. siRNA4, which had the strongest inhibitory effect, reduced PLK1 mRNA in MCF-7 cells by 70% and PLK1 protein in MCF-7 cells by 95% 24 hours after transfection. Cell proliferation was reduced by between 66% and 99% 48 hours after transfection, and apoptosis was increased from 1%–5% to 13%–50% in transfected cells. Transfected SW-480 cells were mitotically arrested, and their centrosomes had lost the ability to nucleate microtubules. HMECs took up siRNAs less efficiently than cancer cells, and transfection with siRNAs targeted to PLK1 did not inhibit their proliferation. Conclusions: PLK1 function appears to be essential for centrosome-mediated microtubule events and, consequently, for spindle assembly. siRNAs targeted against human PLK1 may be valuable tools as antiproliferative agents that display activity against a broad spectrum of neoplastic cells at very low doses.