F (Factor) VIIIa binds to phospholipid membranes during formation of the FXase complex. Free thiols from cysteine residues of isolated FVIIIa A1 and A2 subunits and the A3 domain of the A3C1C2 subunit were labelled with PyMPO maleimide {1-(2-maleimidylethyl)-4-[5-(4-methoxyphenyl)-oxazol-2-yl]pyridinium methanesulfonate} or fluorescein (fluorescence donors). Double mutations of the A3 domain (C2000S/T1872C and C2000S/D1828C) were also produced to utilize Cys(1828) and Cys(1872) residues for labelling. Labelled subunits were reacted with complementary non-labelled subunits to reconstitute FVIIIa. Octadecylrhodamine incorporated into phospholipid vesicles was used as an acceptor for distance measurements between FVIII residues and membrane surface by fluorescence resonance energy transfer. The results of the present study indicate that a FVIII axis on a plane that intersects the approximate centre of each domain is orientated with a tilt angle of ~30-50° on the membrane surface. This orientation predicted the existence of contacts mediated by residues 1713-1725 in the A3 domain in addition to a large area of contacts within the C domains. FVIII variants where Arg(1719) or Arg(1721) were mutated to aspartate showed a >40-fold reduction in membrane affinity. These results identify possible orientations for FVIIIa bound to the membrane surface and support a new interaction between the A3 domain and the membrane probably mediated in part by Arg(1719) and Arg(1721).