When spinach thylakoids were treated with pancreatic phospholipase A2, phospholipids were hydrolyzed and the uncoupled non-cyclic electron flow activity (from H2O to NADP+) was progressively inhibited. By removing, under controlled conditions, hydrolysis products (free fatty acids and lysophospholipids) with bovine serum albumin, it was possible to monitor the inhibition of electron flow activity as a function of the extent of phospholipid depletion. Phospholipid depletion in the outer and inner thylakoid membrane was achieved in a stepwise fashion by incubating thylakoids, first at 2°C for 60 min then at 20°C for 70 min. Complete phospholipid depletion (i.e., 56% of phosphatidylcholine and 74% of phosphatidylglycerol or 70% of the total phospholipids) in the outermonolayer led to less than 20% inhibition of the electron flow activity. In contrast, the depletion of the phospholipid populations (i.e., 44% of phosphatidylcholine and 16% of phosphatidylglycerol or 24% of total phospholipids) localized in the inner monolayer caused more than 80% inhibition of the activity. Finally, a third phosphatidylglycerol population (10%), which is presumably localized in the inner monolayer, was not involved in the electron flow activity. During phospholipid hydrolysis, it was verified that the osmotic responsiveness of thylakoids towards sorbitol remained intact, attesting that the access of phospholipase A2 was restricted to the outer surface of thylakoid vesicles. These results point to the nonequivalent role of phospholipids in the inner and outer monolayers of the thylakoid membrane in supporting the uncoupled non-cyclic electron flow activity. Furthermore, it was shown that after complete depletion of both phospholipids in the outer monolayer, a stepwise delocalization of inner phospholipid molecules through transbilayer movement inhibited the activity in a nonlinear fashion. This suggests the occurrence of interactions having different types and strengths between these innerlipids, more particularly between phosphatidylglycerol and othermembrane components.