Killer whales (Orcinus orca), as apex predators, accumulate high levels of persistent organic pollutants (POPs) such as dichlorodiphenyltrichloroethane and its analogs (DDTs) and face their risks at the population level. The assessment of the function of estrogen receptor alpha (ERα) is crucial for evaluating impact of DDTs on killer whale endocrine and reproductive health. However, due to ethical constraints, little is known about the effects of DDTs on the function of killer whale ERα (kwERα). This study aimed to assess kwERα transactivation potencies in response to various DDTs (p,p'-DDT, o,p'-DDT, p,p'-DDD, o,p'-DDD, p,p'-DDE, o,p'-DDE, and p,p'-DDOH) by New Approach Methodologies (NAMs). We constructed an in vitro kwERα-expressed reporter gene assay and measured transactivation potencies of DDTs as the 10 % effective concentration (REC