BACKGROUND: Selenium (Se), Copper (Cu) and Zinc (Zn) are essential trace elements, required for several cellular functions, showcasing toxicity in either insufficient or excessive concentrations. The selenoprotein thioredoxin reductase 1 (TXNRD1) is directly affected by Se availability and here we hypothesized that it may also be affected by high Cu and Zn concentrations. METHODS AND RESULTS: Using an optimized protocol for the highly selective TXNRD1 activity probe, RX1, we discovered a direct inhibitory effect of Zn on the intracellular TXNRD1 activity, using two different commonly used human cancer cell lines, A549 lung carcinoma and HeLa cervical carcinoma cells. Subsequently, after initial inhibition by Zn, the TXNRD1 activity recovered in both cell lines, in HeLa cells concomitantly with activation of the redox regulatory transcription factor NRF2. High extracellular Cu concentrations did not induce an immediate decrease of intracellular TXNRD1 activity, but decreased its activity upon long-term exposure. While the expression levels of TXNRD1 did not change upon long-term Cu exposure, the selenoprotein glutathione peroxidase 1 (GPX1), that is more dependent upon selenocysteine incorporation, was downregulated, suggesting that higher Cu exposure generally impaired selenoprotein synthesis. CONCLUSION: Our findings support the importance of understanding trace element exposure and availability in basic research, especially in redox biology research, as well as considering Cu and Zn as potential modulators of the cellular capacity of the thioredoxin system and other selenoproteins.