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Objectives A small or a large cavum septi pellucidi (CSP) during routine second trimester sonography may suggest abnormal cerebral development. Therefore, determination of CSP volume with three-dimensional (3D) ultrasound can be valuable. For this purpose, we sought to evaluate the reference ranges and measurement reliability of CSP volume by Virtual Organ Computer-aided AnaLysis (VOCAL). Methods VOCAL software was used to calculate the CSP volume from transabdominal multiplanar datasets of 99 structurally normal fetal ultrasound examinations between 19 and 24 weeks of gestation. Linear regression was utilized to determine reference intervals for CSP volumes as a function of gestational week (GW). Agreement among three evaluators with different proficiency levels (obstetrics and gynecology resident, perinatology fellow, and perinatologist) was assessed, using intraclass correlation coefficients (ICC) and 95% confidence intervals (CI). Results CSP volume and gestational age was positively correlated (r2=0.383, p=0.0001), represented by the following equation: 0.058–(1.016 x GW). Interobserver agreement between perinatologist and fellow was relatively high (ICC, 0.78; 95% CI, 0.70–0.85), whereas limited ultrasound experience (resident) was associated with fair agreement with non-novice observers (ICC for resident and perinatologist, 0.50; 95% CI, 0.29–0.65 and ICC for resident and fellow, 0.57; 95% CI, 0.38–0.71). Conclusions Reference ranges of CSP volumes using VOCAL from 19 0/6 through 24 6/7 weeks of gestation were established. A first-degree model to estimate CSP volume as a function of gestational age was also constructed. CSP volumetry seems reliable when evaluated by an examiner with particular 3D sonography experience.