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Recent data indicate that urban air harbors a complex mixture of industrial chemicals, pesticides, and combustion by-products, many of which may act as endocrine-disrupting chemicals (EDCs). By interfering with the endocrine system, EDCs can affect human health. Hence, there is an urgent need to better characterize outdoor airborne chemical mixtures and their health impact. This study assessed the bioactivity on key targets of EDCs for chemical extracts of the atmospheric vapour phase (i.e., excluding particulate matter) collected from 40 sites across Montreal, Canada, using passive air samplers deployed over 82 summer days. Seven validated bioassays were used to test the ability of each extract to alter the estrogen, androgen, thyroid, and steroidogenesis pathways. Of the 42 urban air extracts tested, none induced alteration of the human thyroid peroxidase activity or activated the human androgen receptor (hAR) but three induced estrogen receptor activation and five inhibited the sodium/iodide symporter by 40-60 %. More than 20 extracts antagonized hESR1 and/or hAR, a few with very strong potency. Yet, blanks used as controls also induced signals in the antagonist mode of the transactivation assays and affected testosterone production in the H295R steroidogenic assay, challenging data interpretations. Overall, our data indicate the potential for thyroid, estrogenic, antiestrogenic and antiandrogenic disruption caused by the chemical mixtures present in the outdoor air of a major metropolis. This work provides one of the first integrated assessments of endocrine activity from atmospheric vapour phase chemical extracts, underscoring the importance of incorporating bioanalytical tools into air-quality and health-risk evaluation frameworks.