The main cofactors involved in Photosystem II (PSII) oxygen evolution activity are borne by two proteins, D1 (PsbA) and D2 (PsbD). In Thermosynechococcus elongatus, a thermophilic cyanobacterium, the D1 protein is predominantly encoded by either the psbA 1 or the psbA 3 gene, the expression of which depends on the environmental conditions. In this work, the Q B site properties in PsbA1-PSII and PsbA3-PSII were probed through the binding properties of DCMU, a urea-type herbicide, and bromoxynil, a phenolic-type herbicide. This was done by using helium temperature EPR spectroscopy and by monitoring the time-resolved changes of the redox state of Q A by absorption spectroscopy in PSII purified from a His 6-tagged WT strain expressing PsbA1 or from a His 6-tagged strain in which both the psbA 1 and psbA 2 genes have been deleted and which therefore only express PsbA3. It is shown that, in both PsbA1-PSII and PsbA3-PSII, bromoxynil does not bind to PSII when Q B is in its semiquinone state which indicates a much lower affinity for PSII when Q A is in its semiquinone state than when it is in its oxidized state. This is consistent with the midpoint potential of Q A − /Q A being more negative in the presence of bromoxynil than in its absence [Krieger-Liszkay and Rutherford, Biochemistry 37 (1998) 17339–17344]. The addition in the dark of DCMU, but not that of bromoxynil, to PSII with a secondary electron acceptor in the Q B − state induces the oxidation of the non-heme iron in a fraction of PsbA3-PSII but not in PsbA1-PSII. These results are explained as follows: i) bromoxynil has a lower affinity for PSII with the non-heme iron oxidized than DCMU therefore, ii) the midpoint potential of the Fe II/Fe III couple is lower with DCMU bound than with bromoxynil bound in PsbA3-PSII; and iii) the midpoint potential of the Fe II/Fe III couple is higher in PsbA1-PSII than in PsbA3-PSII. The observation of DCMU-induced oxidation of the non-heme iron leads us to propose that Q 2, an electron acceptor identified by Joliot and Joliot [FEBS Lett. 134 (1981) 155–158], is the non-heme iron.