The Xenopus Eleutheroembryonic Thyroid Assay (XETA) was recently published as an OECD Test Guideline for detecting chemicals acting on the thyroid axis. However, the OECD validation did not cover all mechanisms that can potentially be detected by the XETA. This study was therefore initiated to investigate and consolidate the applicability domain of the XETA regarding the following mechanisms: thyroid hormone receptor (THR) agonism, sodium-iodide symporter (NIS) inhibition, thyroperoxidase (TPO) inhibition, deiodinase (DIO) inhibition, glucocorticoid receptor (GR) agonism, and uridine 5′-diphospho-glucuronosyltransferase (UDPGT) induction. In total, 22 chemicals identified as thyroid-active or -inactive in Amphibian Metamorphosis Assays (AMAs) were tested using the XETA OECD Test Guideline. The comparison showed that both assays are highly concordant in identifying chemicals with mechanisms of action related to THR agonism, DIO inhibition, and GR agonism. They also consistently identified the UDPGT inducers as thyroid inactive. NIS inhibition, investigated using sodium perchlorate, was not detected in the XETA. TPO inhibition requires further mechanistic investigations as the reference chemicals tested resulted in opposing response directions in the XETA and AMA. This study contributes refining the applicability domain of the XETA, thereby helping to clarify the conditions where it can be used as an ethical alternative to the AMA. •The Xenopus Eleutheroembryonic Thyroid Assay (XETA) was recently developed to screen for thyroid activity of chemicals.•XETA results were compared to Amphibian Metamorphosis Assay (AMA) outcomes for the detection of thyroid-active chemicals.•Results were concordant for all selected mechanisms, but the XETA did not detect inhibition of the sodium-iodide symporter.•Thyroperoxidase inhibition requires further mechanistic investigations due to equivocal responses in the XETA.•The XETA and the AMA consistently identified uridine 5′-diphospho-glucuronosyltransferase inducers as thyroid-inactive.