SummaryWe characterized a set of Arabidopsis mutants deficient in specific light‐harvesting proteins, using freeze‐fracture electron microscopy to probe the organization of complexes in the membrane and confocal fluorescence recovery after photobleaching to probe the dynamics of thylakoid membranes within intact chloroplasts. The same methods were used to characterize mutants lacking or over‐expressing PsbS, a protein related to light‐harvesting complexes that appears to play a role in regulation of photosynthetic light harvesting. We found that changes in the complement of light‐harvesting complexes and PsbS have striking effects on the photosystem II macrostructure, and that these effects correlate with changes in the mobility of chlorophyll proteins within the thylakoid membrane. The mobility of chlorophyll proteins was found to correlate with the extent of photoprotective non‐photochemical quenching, consistent with the idea that non‐photochemical quenching involves extensive re‐organization of complexes in the membrane. We suggest that a key feature of the physiological function of PsbS is to decrease the formation of ordered semi‐crystalline arrays of photosystem II in the low‐light state. Thus the presence of PsbS leads to an increase in the fluidity of the membrane, accelerating the re‐organization of the photosystem II macrostructure that is necessary for induction of non‐photochemical quenching.