The umbilical artery, aorta, and middle cerebral artery pulsatility indices were investigated by pulsed Doppler ultrasound in 73 fetuses at 18–37 weeks of gestation, before and after fetal blood sampling performed either at the placental cord insertion (n = 46) or at the intrahepatic vein (n = 27). At the end of the procedure, after randomization, 35 fetuses were infused amounts of normal saline equal to the blood volume withdrawn, and 38 fetuses served as controls. Following blood sampling, the umbilical artery pulsatility indices decreased both in controls (p = 0.004) and in the saline group (p = 0.006). The middle cerebral artery velocity waveforms exhibited similar changes only in controls (p = 0.01), and no changes in fetal heart rate and aortic pulsatility indices were recorded in either group. The changes in blood flow velocity waveforms did not correlate with gestational age and the blood volume sampled, and were similar whether the site of sampling was the placental cord insertion or the intrahepatic vein. In 10 acidemic and/or hypoxemic fetuses, pulsatility indices in the umbilical and middle cerebral arteries were not modified by the blood sampling procedure. The release of vasoactive substances is most likely the cause of diminished vascular resistances following fetal blood sampling. Hypoxemic/acidemic fetuses may fail to mount a normal vasodilative response to needle punctur