AbstractThe effects of DCMU and NaN3were studied on menadione‐mediated photophosphorylation in broken spinach chloroplasts kept in low oxygen tension in Tricine or HEPES buffers at either high or reduced irradiances. – (A) At high irradiance (131 W. m−2) andabsenceof NaN3the ATP formation was inhibited by DCMU regardless of the type of buffer used. – (B) At high irradiance andpresenceof NaN3some concentrations of DCMU stimulated, whilst others inhibited the ATP formation in a HEPES buffer. The ATP formation was predominantly inhibited by DCMU in a Tricine buffer. – (C) At reduced irradiance (57 W. m−2) the chloroplasts in a HEPES buffer were almost insensitive towards DCMU both in the presence and absence of NaN3. – (D) Chloroplasts in a Tricine buffer were slightly stimulated in their ATP formation by DCMU at reduced irradiance either with or without the presence of NaN3in the experimental medium. When menadione acts as a terminal electron acceptor, oxygen is consumed on its reoxidation. The results indicate that this process may occur with oxygen released by the splitting of water as the main oxidant. – The data also demonstrate the importance of caution when selecting buffering substances as well as when choosing light intensities for experiments on photophosphorylation