ATP8A2 is a P(4)-ATPase ("flippase") located in membranes of retinal photoreceptors, brain cells, and testis, where it mediates transport of aminophospholipids toward the cytoplasmic leaflet. It has long been an enigma whether the mechanism of P(4)-ATPases resembles that of the well-characterized cation-transporting P-type ATPases, and it is unknown whether the flippases interact directly with the lipid and with counterions. Our results demonstrate that ATP8A2 forms a phosphoenzyme intermediate at the conserved aspartate (Asp(416)) in the P-type ATPase signature sequence and exists in E(1)P and E(2)P forms similar to the archetypical P-type ATPases. Using the properties of the phosphoenzyme, the partial reaction steps of the transport cycle were examined, and the roles of conserved residues Asp(196), Glu(198), Lys(873), and Asn(874) in the transport mechanism were elucidated. The former two residues in the A-domain T/D-G-E-S/T motif are involved in catalysis of E(2)P dephosphorylation, the glutamate being essential. Transported aminophospholipids activate the dephosphorylation similar to K(+) activation of dephosphorylation in Na(+),K(+)-ATPase. Lys(873) mutants (particularly K873A and K873E) display a markedly reduced sensitivity to aminophospholipids. Hence, Lys(873), located in transmembrane segment M5 at a "hot spot" for cation binding in Ca(2+)-ATPase and Na(+),K(+)-ATPase, appears to participate directly in aminophospholipid binding or to mediate a crucial interaction within the ATP8A2-CDC50 complex. By contrast, Lys(865) is unimportant for aminophospholipid sensitivity. Binding of Na(+), H(+), K(+), Cl(-), or Ca(2+) to the E(1) form as a counterion is not required for activation of phosphorylation from ATP. Therefore, phospholipids could be the only substrate transported by ATP8A2.