Objectives The aims of this prospective screening study were to calculate the success rate in measuring fetal nuchal translucency thickness and to assess the intra- and interoperator variabilities of the measurement using three-dimensional ultrasound. Subjects and methods Nuchal translucency thickness was measured prospectively at 10-14 weeks of gestation in 616 singleton pregnancies in an unselected population from November 1997 to June 1999. Three-dimensional transvaginal ultrasound was used in all cases. A new procedure of nuchal translucency thickness measurement was developed from the volumetric data of the fetal neck. In 86 pregnancies, the intra- and interoperator variabilities were examined. Results In all cases, we were able to obtain good sagittal views of fetuses and to measure the nuchal translucency thickness. Out of 616 fetuses, 15 were classified as screen-positive, having increased nuchal translucency thickness of greater than or equal to 3 mm. Eight of these fetuses underwent karyotyping. Two cases were confirmed with Down's syndrome and two with Turner's syndrome. Two others were spontaneously aborted before invasive testing. The intra- and interoperator variabilities were satisfactory. In addition, patients felt negligible discomfort, because of the short examination time and the ease with which the operators performed the measurement, owing to the procedure being well established. Conclusion Three-dimensional ultrasound improved fetal nuchal translucency thickness measurement at 10-14 weeks of gestation, but its advantages may need further evaluation.