OBJECTIVES: Accuracy of estradiol measurements is important but conventional proficiency testing (PT) cannot assess accuracy when possibly non-commutable samples are used and method peer-group means are the targets. Accuracy-based assessment of estradiol measurements is needed. Design and Methods Five serum samples were prepared from single donors, frozen, and distributed overnight to 76 New York State Department of Health (NYSDOH)-certified laboratories. Participants analyzed samples for estradiol. The biases of group means were assessed against the Centers for Disease Control and Prevention (CDC)-defined targets, evaluated using the Hormones Standardization Program (HoSt) E2 performance criterion of ±12.5%. Each laboratory's performance was evaluated using total allowable error (acceptance limits) of target ±25% or ±15 pg/mL (55.1 pmol/L) (whichever was greater, NYSDOH), target ±30% (Clinical Laboratory Improvement Amendments [CLIA]), and target ±26% (minimal limit based on biological variation [BV]). RESULTS: The biases (range) were 34% (-17% to 175%), 40% (-33% to 386%), 16% (-45% to 193%), 5% (-27% to 117%), and -4% (-31% to 21%), for samples at estradiol of 24.1, 28.4, 61.7, 94.1 and 127 pg/mL, or 88.5, 104.3, 226.5, 245.4 and 466.2 pmol/L, respectively. Large positive method/analytical systematic biases were revealed for 9 commonly used method/analytical systems in the United States at low estradiol concentrations. Of the 9 analytical systems, 0, 0, 3, 7 and 6 met the HoSt criterion for the samples with estradiol at the five respective concentrations. PT evaluation showed that 59%, 69% and 87% of laboratories would receive a PT event pass score when the CDC-defined target and a criterion of NYSDOH, CLIA or BV was used, respectively. However, >95% laboratories would obtain PT pass score if method peer-group means were used as targets regardless of the criterion used. CONCLUSIONS: Improvement in accuracy of estradiol measurements is needed, particularly at low estradiol concentrations. Accuracy-based PT provides unambiguous information about the accuracy of methods/analytical systems.