In screening for epithelial ovarian cancer, unnecessary surgery can be reduced by limiting use of transvaginal ultrasound (TVU) to women with increasing CA125 serum levels. Replacing or augmenting TVU with measurement of a serum marker specific for malignancy might further improve screening performance. Serum samples from 112 invasive ovarian cancer patients and 706 matched control subjects from the Prostate, Lung, Colorectal, and Ovarian trial were used to evaluate human epididymis protein 4 (HE4), mesothelin, matrix metalloproteinase 7 (MMP7), SLPI, Spondin2, and insulin-like growth factor binding protein 2 (IGFBP2) for their potential use in screening. TVU results were available for a subset of 84 patients and 516 control subjects used to compare the best marker with TVU. HE4 was found to perform better than TVU as a second-line screen, confirming 27 of 39 cancers with increasing CA125 serum levels compared with 17 cancers confirmed by TVU (P = .03). Serum HE4 levels were found to increase with age and smoking status, suggesting that a longitudinal algorithm might improve its performance.