This study investigated the appropriate tourniquet pressure (TP) and duration of tourniquet application for venipuncture by calculating the venous cross-section (VCS) area on ultrasonography. Twenty healthy volunteers without cardiovascular risk factors were enrolled in this study. A target vein (either a cephalic or median cubital vein) was selected on ultrasonography. The pneumatic tourniquet was inflated using a rapid cuff inflator system at setting pressure for 120 sec. TP strength was varied from 20 mmHg to 100 mmHg, in 20 mmHg increments. The order of TP was randomized. Comparisons among more than 3 groups were performed by one-way repeated-measures ANOVA and the Bonferroni method. The VCS area increased rapidly until 10 sec after tourniquet inflation. The VCS area then increased gradually until 30 sec after tourniquet inflation. After that, the VCS area did not increase remarkably. The VCS area increased with TP strength up to 80 mmHg, but the VCS area at TP 100 mmHg decreased to less than that at TP 40 mmHg. Based on these results, we recommend a tourniquet pressure of 60 mmHg, and duration of tourniquet application is 30 to 60 sec for venipuncture.