Kinetic studies of protein dephosphorylation in photosynthetic thylakoid membranes revealed specifically accelerated dephosphorylation of photosystem II (PSII) core proteins at elevated temperatures. Raising the temperature from 22 degrees C to 42 degrees C resulted in a more than 10-fold increase in the dephosphorylation rates of the PSII reaction center proteins D1 and D2 and of the chlorophyll a binding protein CP43 in isolated spinach (Spinacia oleracea) thylakoids. In contrast the dephosphorylation rates of the light harvesting protein complex and the 9-kD protein of the PSII (PsbH) were accelerated only 2- to 3-fold. The use of a phospho-threonine antibody to measure in vivo phosphorylation levels in spinach leaves revealed a more than 20-fold acceleration in D1, D2, and CP43 dephosphorylation induced by abrupt elevation of temperature, but no increase in light harvesting protein complex dephosphorylation. This rapid dephosphorylation is catalyzed by a PSII-specific, intrinsic membrane protein phosphatase. Phosphatase assays, using intact thylakoids, solubilized membranes, and the isolated enzyme, revealed that the temperature-induced lateral migration of PSII to the stroma-exposed thylakoids only partially contributed to the rapid increase in the dephosphorylation rate. Significant activation of the phosphatase coincided with the temperature-induced release of TLP40 from the membrane into thylakoid lumen. TLP40 is a peptidyl-prolyl cis-trans isomerase, which acts as a regulatory subunit of the membrane phosphatase. Thus dissociation of TLP40 caused by an abrupt elevation in temperature and activation of the membrane protein phosphatase are suggested to trigger accelerated repair of photodamaged PSII and to operate as possible early signals initiating other heat shock responses in chloroplasts.