Wheat seedlings (Triticum aestivum L.) develop plastids (etioplasts and chloroplasts) which exhibit alterations in inner membrane organisation after treatment with Norflurazon (NF), an inhibitor of carotenoid biosynthesis. In dark-grown plants, it results in a decreased amount of partitions (contact zones) between prothylakoids. Under weak red radiation (WRR), plants contain chloroplasts devoid of grana. Using the fluorescent probe 9-amino acridine (9-AA), the average surface charge density of isolated prothylakoids (PTs) was -21.8±3.2 mC m-2 and -27.4±2.6 mC m-2 in the control and after treatment, respectively. Thylakoid membranes isolated from plants grown under WRR exhibited slightly more negative values, -23.5±2.9 mC m-2 and -29.0±2.1 mC m-2, in control and after NF treatment, respectively. The surface charge density of de-stacked thylakoids from greenhouse-grown untreated plants, containing extensive grana stacking, was -34.3±2.5 mC m-2. Assays using the fluorescent probe of DPH (1,6-diphenyl-1,3,5-hexatriene) showed a higher polarisation value when incorporated into thylakoids from NF-treated plants compared to untreated plants grown under WRR. The highest polarisation value was found in untreated plants grown in the greenhouse. This indicates a lower rotation transition of the probe in the lipid environment of thylakoids after NF treatment, which can be interpreted as more rigid membranes. Hence the surface charge density and the mobility of membrane components may play a major role for the formation of partitions in dark-grown plants and in the formation of grana in plants grown under WRR.