The efficiency of small interfering RNA (siRNA)-induced gene knockdown is hampered by low transfection efficiency. We established a novel and simple double transfection method using specific siRNA duplexes targeted against human thrombin receptor PAR-1 in DU 145 prostate cancer cells. The initial siRNA transfection of cell suspensions followed by re-transfection of adherent cells on the following day resulted in undetectable PAR-1 mRNA and absent receptor protein. PAR-1 mRNA expression was silenced for up to five days. Functional studies showed that PAR-1 gene silencing in DU 145 cells abolished the modulating effects of thrombin on cell adhesion to the extracellular matrix proteins, fibronectin and laminin, thus demonstrating the essential role of PAR-1 in mediating thrombin effects on DU 145 cell adhesion.