The effect of protein phosphorylation on the distribution of chlorophyll-protein complexes between appressed and non-appressed thylakoid regions of spinach chloroplasts has been investigated. Stroma lamellae vesicles and inside-out vesicles, representative of non-appressed and appressed thylakoids, respectively, were isolated from thylakoid membranes before and after phosphorylation. The fractions were analyzed with respect to the yield of vesicles, incorporation of [32P]phosphate into the light-harvesting chlorophyll-a / b—protein complex, chlorophyll composition, and the relative content of the main chlorophyll-protein complexes. The yield of inside-out vesicles from phosphorylated thylakoids was 20% lower than that from control thylakoids, indicating a partial destacking. The specific incorporation of [32P]phosphate into the light-harvesting chlorphyll-a / b—protein complex was at least four times higher in stroma lamellae vesicles than in inside-out vesicles. The proportion of the light-harvesting chlorophyll-a / b—protein complex in stroma lamellae vesicles increased from 13% to 21% of their total chlorphyll after phosphorylation of the thylakoids. There was also a corresponding increase in the level of chlorophyll b. These observations strongly suggest that phosphorylated light-harvesting complexes migrate from appressed thylakoids rich in photosystem 2 to non-appressed thylakoids rich in photosystem 1. In contrast, there was no evidence for a lateral migration of the chlorophyll-a—protein complex of photosystem 2 after phosphorylation. Our results indicate that a lateral migration of phosphorylated light-harvesting complexes in combination with a lateral separation of photosystem 1 and 2 to the different thylakoid regions, is a basis for the regulation of excitation energy between the two photosystems.