Higher plant thylakoid membranes contain a protein kinase that phosphorylates certain threonine residues of light-harvesting complex II (LHCII), the main light-harvesting antenna complexes of photosystem II (PSII) and some other phosphoproteins (Allen, Biochim Biophys Acta 1098:275, 1992). While it has been established that phosphorylation induces a conformational change of LHCII and also brings about changes in the lateral organization of the thylakoid membrane, it is not clear how phosphorylation affects the dynamic architecture of the thylakoid membranes. In order to contribute to the elucidation of this complex question, we have investigated the effect of duroquinol-induced phosphorylation on the membrane ultrastructure and the thermal and light stability of the chiral macrodomains and of the trimeric organization of LHCII. As shown by small angle neutron scattering on thylakoid membranes, duroquinol treatment induced a moderate (~10%) increase in the repeat distance of stroma membranes, and phosphorylation caused an additional loss of the scattering intensity, which is probably associated with the partial unstacking of the granum membranes. Circular dichroism (CD) measurements also revealed only minor changes in the chiral macro-organization of the complexes and in the oligomerization state of LHCII. However, temperature dependences of characteristic CD bands showed that phosphorylation significantly decreased the thermal stability of the chiral macrodomains in phosphorylated compared to the non-phosphorylated samples (in leaves and isolated thylakoid membranes, from 48.3 degrees C to 42.6 degrees C and from 47.5 degrees C to 44.3 degrees C, respectively). As shown by non-denaturing PAGE of thylakoid membranes and CD spectroscopy on EDTA washed membranes, phosphorylation decreased by about 5 degrees C, the trimer-to-monomer transition temperature of LHCII. It also enhanced the light-induced disassembly of the chiral macrodomains and the monomerization of the LHCII trimers at 25 degrees C. These data strongly suggest that phosphorylation of the membranes considerably facilitates the heat- and light-inducible reorganizations in the thylakoid membranes and thus enhances the structural flexibility of the membrane architecture.