Per- and polyfluoroalkyl substances (PFAS) are a group of xenobiotics that are widely distributed throughout the aquatic environment. Many PFAS are possible thyroid hormone (TH) system disrupting compounds, because they have the capacity to -amongst other- inhibit the TH thyroxine (T4) from binding to its transport protein transthyretin (TTR). This study investigated the occurrence of TH-displacing activity in the Dutch water cycle, and more specifically, the contribution of PFAS to this effect. Over one year of monitoring data of 29 PFAS (linear and branched) showed the continuous presence of PFAS in drinking waters and their surface water sources. Secondly, the FITC-T4 and TTR-TRβ-CALUX bioassays were mutually compared using positive (HPLC-grade water spiked with PFOA) and negative control samples (HPLC-grade water), as well as relative potency factors (RPFs) of up to 20 PFAS congeners. Both assays were found to be suitable for measuring TH-displacing activity in water samples. As a third aim, a field study was performed in the Dutch water cycle that was comprised of samples from drinking water, surface water, PFAS contaminated sites, and 2 wastewater treatment plants. All samples were analyzed with 1. chemical analysis for 29 PFAS, 2. the FITC-T4 bioassay, and 3. the TTR-TRβ-CALUX bioassay. The bioassays mutually showed good correlation (R2 0.85). Bioanalytical equivalent concentrations (BEQ) based on chemically-determined concentrations and RPFs (BEQchem) revealed that analyzed PFAS only explained ≤4.1 % of their activity in water extracts measured by both bioassays (BEQbio). This indicated that as yet unknown compounds contribute to the majority of the measured TH-displacing activity. Moreover, water treatment processes (e.g. DW production from SW) showed a larger contribution of target PFAS to the BEQbio. This could be a first lead to identify unknown compounds that contribute to this activity, and as such, enable the assessment of possible risks associated by the occurrence of TH-displacing activity in ...