Objectives Three-dimensional (3D) power Doppler ultrasonography provides indices to quantify moving blood within a volume of interest (e.g. ovary, endometrium, tumor or placenta). The purpose of this study was to determine the influence of ultrasound instrument settings on vascularization index (VI) and flow index (FI) at different flow velocities, using a specially built flow phantom with a small tube diameter. Methods Blood-mimicking fluid was pumped at 10-100 mL/h through a plastic tube with a diameter of 0.65 mm within a virtual spherical volume (content 137.12 cm(3)) of a Voluson 730 Expert 3D power Doppler ultrasound instrument. VI and FI were determined at different pulse repetition frequency (PRF) settings, with minimal and maximal wall motion filter (WMF) settings. The measured VI was compared with the actual VI. Results The ability to measure VI and FI at different flow velocities was highly dependent on the PRF and WMF settings. In our experimental set-up, using a PR F of 03 kHz, flow velocities of about 2 cm/s and higher could be registered. Measured VI was overestimated up to 44 times relative to actual VI. Conclusions Our main finding in a laboratory set-up was a considerable overestimation of moving blood volume using 3D power Doppler ultrasound in a single small tube. The degree of overestimation depends on The spatial resolution and on the settings of the ultrasound instrument. When small vessels are involved in a clinical setting, interpretation of VI should take this overestimation of moving blood volume into account. Copyright (C) 2008 ISUOG. Published by John Wiley & Sons, Ltd.