Structural analysis of weakly ordered two-dimensional (2-D) ordered arrays of Photosystem II (a photosynthetic membrane protein complex) has been carried out using single-particle alignment and averaging (SPA). Although this averaging technique is normally applied to randomly oriented solubilised particles, we have found that it is also highly effective for the analysis of membrane-located 2-D ordered arrays and that the arrays can be used to check the effectiveness of the rotational alignment routines. Successful rotational alignment varied in the range 75 to 66% and 26 to 19%, depending on the stain used and hence the signal-to-noise ratio, and the data were used to address questions of space group assignments for Photosystem II 2-D ordered arrays. We thus hoped to settle the controversy as to the oligomeric form of PS II in native thylakoids, i.e., the monomeric vs. dimeric model. The methods were applied to the study of the removal and rebinding of extrinsic subunits in Photosystem II. Information on the location of the subunits in this large membrane protein complex is presented here.