OBJECTIVE - To compare the accuracy measures of the random glucose test and the 50-g glucose challenge test as screening tests for gestational diabetes mellitus (GDM). RESEARCH DESIGN AND METHODS - In this prospective cohort study, pregnant women without preexisting diabetes in two perinatal centers in the Netherlands underwent a random glucose test and a 50-g glucose challenge test between 24 and 28 weeks of gestation. If one of the screening tests exceeded predefined threshold values, the 75-g oral glucose tolerance test (OGTT) was performed within 1 week. Furthermore, the OGTT was performed in a random sample of women in whom both screening tests were normal. GDM was considered present when the OGTT (reference test) exceeded predefined threshold values. Receiver operating characteristic (ROC) analysis was used to evaluate the performance of the two screening tests. The results were corrected for verification bias. RESULTS - We included 1,301 women. The OGTT was performed in 322 women. After correction for verification bias, the random glucose test showed an area under the ROC curve of 0.69 (95% CI 0.61-0.78), whereas the glucose challenge test had an area under the curve of 0.88 (0.83-0.93). There was a significant difference in area under the curve of the two tests of 0.19 (0.11-0.27) in favor of the 50-g glucose challenge test. CONCLUSIONS - In screening for GDM, the 50-g glucose challenge test is more useful than the random glucose test.