Manganese seems to be a key component of the photosynthetic O2 evolving apparatus. This conclusion stems from a number of observations including Mn deficiency studies with algal cultures and experiments involving various washing procedures of isolated thylakoid membranes [ 1,2]. A dogma has been established from these observations that there exists a mangano-protein responsible for the binding and subsequent oxidation of water to molecular oxygen. The water-binding protein is thought to contain either 2 or 4 Mn atoms in its catalytic centre. This general idea has been used to interpret experiments using NMR and EPRspectroscopy [3,4]. Although the concept of a mangano-protein being involved in the water oxidation process is intuitively attractive, the evidence for its existence is poor [S]. Claims that a 65 000-M, protein, containing 2 Mn atoms, is intimately involved in O2 evolution and can be readily isolated from thylakoid membranes [6] have not been substantiated [7]. One of the most important indirect observations which support the idea that a mangano-protein is involved in O2 evolution is the effect of washing isolated thylakoid membranes with alkaline Tris (hydroxymethyl) amino methane (Tris) buffer. Such a treatment usually removes -2/3rd of the total Mn content of the membranes and inhibits photosynthetic O2 evolution [ 1,2]. From this result it has been concluded that the Trisremovable Mn is derived from the water-binding protein and that this protein contains 4 Mn atoms [ 11. No functional role has been suggested for the firmly bound Mn not removed by the Tris wash. The idea that alkaline Tris washing inhibits O2 evolution because it removes Mn atoms from the water binding protein is widely assumed and, indeed, this washing procedure is often used as a test of reliability that a spectroscopic signal is indicative of Mn functionally active in the water splitting process [8]. However, there are doubts to the importance of Tris-removable Mn in O2 evolution which stem from [9-l 11: After alkaline Tris washing of spinach thylakoids, O2 evolution could be restored by treatments such as ‘dark reactivation’ involving simply addition of reduced dichlorophenol indophenol (DCIP) or hydroquinone (HQ) to Tris-washed membranes [9]. Under the best conditions, -70% of the original O2 evolving capacity could be restored even though the Mn content of the membranes had been significantly reduced [9,10]. Such a result is inconsistent with the view that Tris washing removes functionally active Mn from the water splitting protein. An explanation for this inconsistency has come from [12,13]. Using EPR and atomic absorption spectroscopy to detect Mn2+ and total Mn, respectively, it was concluded that Tris washing removed only 10% of the total Mn and that 60% of the Mn pool was released into the intrathylakoid space as a consequence of the treatment [ 121. Further, the dark reactivation procedure may be brought about simply by a reinsertion of this free Mn trapped within the intrathylakoid space into the membrane-bound protein responsible for water oxidation [ 131. Here we show, using the dark reactivation procedure, that it is possible to restore O2 evolution from Tris-treated inside-out thylakoid membranes. Our results indicate that the Tris-removable Mn is not necessary for photosynthetic O2 evolution and that if a mangano-protein is involved in the water oxidation process it must be associated with the firmly bound Mn pool not removed by alkaline Tris treatment.