In previous publications, we have shown, by using spin-labeled derivatives, that the translocation of phosphatidylserine and phosphatidylethanolamine from the outer to the inner monolayer of human erythrocyte membrane is a protein-mediated phenomenon, which requires hydrolisable Mg²⁺-ATP. The inhibition by intracellular Ca²⁺ (0.2 μM) or by extracellularly added vanadate (50 μM) was reported (Seigneuret, M. and Devaux, P.F. (1984) Proc. Natl. Acad. Sci. USA 81, 3751–3755; Zachowski, A., Favre, E., Cribier, S., Hervé, P. and Devaux, P.F. (1986) Biochemistry 25, 2585–2590). The present article gives further insight into the effects of intracellular and extracellular ions on the aminophospholipid translocation in human erythrocytes. By measuring the cell ATP concentration, we now show that the inhibitory effect of intracellular calcium on spin-labeled aminophospholipid translocation is partly due to the ATP depletion, which follows the increased consumption by the calcium pump. However, a direct inhibitory effect of cytosolic Ca²⁺ on the aminophospholipid translocase can be demonstrated by measuring the initial rate of aminophospholipid translocation in the presence of variable amounts of intracellular calcium, at fixed ATP concentrations. Moreover, the transmembrane equilibrium distribution of phosphatidylserine and phosphatidylethanolamine are affected differently by Ca²⁺: when cytosolic Ca²⁺ concentration is increased, alteration of phosphatidylethanolamine distribution begins as soon as the inward translocation is affected by Ca²⁺ (approx. 50 nM), whereas phosphatidylserine distribution remains unchanged within a large inhibitory range of cytosolic Ca²⁺ concentrations and decreases above 0.2 μM of free Ca²⁺ within the cytosol. Decrease of the intracellular Mg²⁺ concentration below its physiological value (approx. 2 mM) results in the inhibition of aminophospholipid inward transport, whereas increase of Mg²⁺ concentration does not modify this transport. If Mn²⁺ is substituted for Mg²⁺, part of the aminophospholipid translocation is maintained, whereas if Co²⁺ is substituted for Mg²⁺, the rapid translocation is completely abolished. Concentrations as high as a millimolar of extracellular Ca²⁺, Mg²⁺ or Mn²⁺ have no effect on the aminophospholipid translocation. The less usual cations Cr³⁺, Fe²⁺, Cu²⁺, Sn²⁺ and Eu³⁺ are also uneffective. With extracellular Ni²⁺ or Co²⁺, some inhibition can be observed, half inhibition by Ni²⁺ corresponding to 500 μM. Vanadyl (VO²⁺), on the other hand, is a potent inhibitor of the aminophospholipid translocation when applied on the extracellular surface, half-inhibition being reached around 30 μM. Finally, the effect of vanadate (VO₄³⁻) was also investigated. Half-inhibition by extracellularly added vanadate was found at 50 μM. However, pretreatment of the cells by a blocker of the anion carrier band 3 partly prevented the inhibitory property of vanadate. This suggests that vanadate effectively acts from the cell interior. Comparison between ionic regulation of the aminophospholipid translocation in human erythrocytes and the influence of ions on cell shape indicates that the asymmetry of phospholipid distribution is probably a major determinant in the control of the normal discoid shape.