Cyclosporine (CsA) is the most widely used immunosuppressive agent for the prevention of acute graft-versus-host disease (GVHD). In a previous report, the incidence of acute GVHD was decreased by increasing the target blood concentration of CsA during a continuous infusion from 300 to 500 ng/mL without excessive toxicities. To confirm these results, we retrospectively analyzed 69 patients who received a continuous infusion of CsA at a higher target CsA level between 450 and 550 ng/mL (CsA500 group) and compared the clinical outcome with 29 patients who received CsA with a lower target concentration between 250 and 350 ng/mL (CsA300 group). The target concentration was determined based on the status of background diseases. Multivariate analysis revealed that the incidence of grade III-IV acute GVHD was significantly lower in the CsA500 group, although the incidence of grade II-IV acute GVHD was not different. Toxicities were equivalently observed between the two groups. Concomitant administration of voriconazole or itraconazole and higher hematocrit were identified as independent significant factors for higher concentration/dose ratio of CsA. The average dose of CsA to maintain CsA level around 500 ng/mL was higher compared with the previous study (3.4 mg/kg vs. 2.7 mg/kg at three wk), probably due to the difference in measuring method of CsA concentration. We conclude that continuous infusion of CsA with a target level between 450 and 550 ng/mL is a feasible and effective GVHD prophylaxis, but caution should be paid for the difference in measuring method.