We aim to test the hypothesis that two-dimensional (2-D) fetal adrenal gland volume (AGV) measurements offer similar volume estimates as volume calculations based on 3-D technique. Fetal AGV was estimated by three-dimensional (3-D) ultrasound (VOCAL) in 93 women with signs/symptoms of preterm labor and 73 controls. Fetal AGV was calculated using an ellipsoid formula derived from 2-D measurements of the same blocks (0.523 × length × width × depth). Comparisons were performed by intraclass correlation coefficient (ICC), coefficient of repeatability, and Bland-Altman method. The corrected AGV (cAGV; AGV/fetal weight) was calculated for both methods and compared for prediction of preterm birth (PTB) within 7 days. Among 168 volumes, there was a significant correlation between 3-D and 2-D methods (ICC = 0.979; 95% confidence interval CI: 0.971 to 0.984). The coefficient of repeatability for the 3-D was superior to the 2-D method (intraobserver 3-D: 30.8, 2-D:57.6; interobserver 3-D:12.2, 2-D: 15.6). Based on 2-D calculations, cAGV ≥ 433 mm3/kg was best for prediction of PTB (sensitivity: 75%, 95% CI = 59 to 87; specificity: 89%, 95% CI = 82 to 94). Sensitivity and specificity for the 3-D cAGV (cutoff ≥ 420 mm3/kg) was 85% (95% CI = 70 to 94) and 95% (95% CI = 90 to 98), respectively. In receiver-operating-curve curve analysis, 3-D cAGV was superior to 2-D cAGV for prediction of PTB (z = 1.99, p = 0.047). 2-D volume estimation of fetal adrenal gland using ellipsoid formula cannot replace 3-D AGV calculations for prediction of PTB.