Hemoglobin vesicles, which encapsulated concentrated hemoglobin (Hb) with a bilayer of dipalmitoylphosphatidylcholine/cholesterol/palmitic acid, were prepared under various preparation conditions in order to decrease the number of bilayers (n) constructing the vesicle and increase the Ho concentration in the interior of the vesicle ([Hb](in)). n decreased when the surface potential of the bilayer became more negative because of the electrostatic repulsion between the bilayers, while with a changing xi-potential of Hb from positive to negative, [Hb](im) showed a precipitous fall because of the electrostatic repulsion between Hb and the surface of the bilayer. A temperature decrease leads to a quality increase in the Hb vesicles ([Hb]/[lipid]) in spite of the [Hb](in) decrease by the viscosity increase of the Hb solution. This is explained by the effective reduction of n due to the reduction in membrane fluidity and the protonation of Hb.