The glycerolipid digalactosyl diacylglycerol (DGDG) is exclusively associated with photosynthetic membranes and thus may play a role in the proper assembly and maintenance of the photosynthetic apparatus. Here we employ a genetic approach based on the dgd1 mutant of Arabidopsis thaliana to investigate the function of DGDG in thylakoid membranes. The primary defect in the genetically well-characterized dgd1 mutant resulted in a 90% reduction of the DGDG content. The mutant showed a decreased photosystem II (PSII) to photosystem I ratio. In vivo room- and low-temperature (77 K) chlorophyll fluorescence measurements with thylakoid preparations are in agreement with a drastically altered excitation energy allocation to the reaction centers. Quantification of pigment-binding apoproteins and pigments supports an altered stoichiometry of individual pigment-protein complexes in the mutant. Most strikingly, an increase in the amount of peripheral light-harvesting complexes of PSII relative to the inner antenna complexes and the PSII reaction center/core complexes was observed. Regardless of the severe alterations in thylakoid organization, photosynthetic oxygen evolution was virtually not compromised in dgd1 mutant leaves.