BACKGROUND: Laboratory diagnosis for ovarian cancer is mostly based on the quantitative determination of CA125. Over the past years, a number of additional markers for ovarian cancer have been proposed and studied. Human epididymis protein 4 (HE4) has accordingly emerged as a new biomarker for the detection of ovarian cancer. To evaluate the new automated HE4 assay, we studied analytical performance, and established reference ranges. METHODS: We evaluated precision performances and linearity of the HE4 assay. We also evaluated reference ranges for HE4 and CA125 according to age. Lastly, we investigated the correlation between HE4 and CA125. RESULTS: The precision study showed excellent results for both high and low control. The 95% upper reference ranges for HE4 and CA125 levels were 81.0 pmol/L (90% confidence interval [CI], 63.0-103.1) and 28.6 U/mL (90% CI, 25.4-36.4), respectively. There was no correlation between HE4 and CA125 (r = -0.002, P = 0.9793) in healthy women. Reference ranges of HE4 tended to be slightly higher for the older groups as compared to the younger groups. CA125 were considerably decreased in the oldest age group (ages 70 to 79). CONCLUSIONS: The new automated HE4 assay showed good analytical performance, age-related variable results and no correlation with CA125. Though further studies for clinical and diagnostic effectiveness of HE4 assay in screening and diagnosing ovarian cancer are needed for routine use of HE4, HE4 in combination with CA125 is likely to be more useful diagnostically than CA125 alone.