Annexin A5 is a soluble protein which binds to negatively charged phospholipids, such as dioleoylphosphatidylserine (DOPS), in the presence of Ca2+. A solid-solid phase transition between two two-dimensional crystal forms of annexin A5, p6 and p3, previously identified by electron microscopy (EM), was studied in situ on supported phospholipid bilayers (SPBs) by atomic force microscopy (AFM) and ex situ on lipid monolayers by EM. The ability to directly vary the protein surface density on SPBs allowed a delicate and systematic way to investigate the reversibility of the transition with the AFM. The p6 crystal form was found to form first and to cover the entire lipid surface available, at all DOPS concentrations studied (5-95%). An increase in protein surface density, achievable only on SPBs (or monolayers) containing sufficient amounts of DOPS, destabilized the p6 phase. The transition to the more compact (p3) form occurred via local melting of the p6 phase at defects, such as grain boundaries, initially present in p6 crystals. On mono- or bilayers with lower DOPS contents, the density-dependent p6-p3 transition was not observed, but the p3 crystal form could still be reached by disturbing the system mechanically.