The fluidity of the hydrophobic interior of phospholipid vesicles after calcium-dependent binding of human annexin V (AVH) was studied using EPR spectroscopy. Vesicles (SUVs) composed of PC or PE and an acidic phospholipid (alternatively PS, PA, or CL) were probed at different bilayer depths by either phosphatidylcholine, or the accompanying acidic phospholipid, bearing a spin label probe at position C-5, C-12, or C-16 of the sn-2 acyl chain. Alternatively, the vesicle surface was probed with a polar head spin labeled PE (PESL). The EPR spectra of annexin-bound bilayer domain(s) were obtained by computer spectral subtraction. The order parameter values (S) from the resulting difference spectra revealed that the bilayer hydrophobic interior has a greatly altered fluidity gradient, with an increased rigidity up to the C-12 position. Thereafter, the rigidification progressively vanished. The effect is not linked to the phospholipid class, since all the acidic phospholipid spectra, as well as phosphatidylcholine, shared the same sensitivity to the bound protein. The observed membrane rigidification appears to parallel the "crystallizing" tendency of vesicle-bound annexin V, but may not be involved in the calcium channeling activity of this protein.