The effect of molecular chirality of alanine molecule on the two dimensional ordering on Cu(001) surface has been studied by high resolution scanning tunneling microscope. It is identified that in the antiphase c(2 x 4) domains on the terraces, two bright spots with different shape, corresponding to each alanine molecule, form chains along [(1) over bar 30] or [(3) over bar 10] direction. Their orientation changes at the domain boundaries along [110] direction from [(1) over bar 30] to [(3) over bar 10] direction in accord with the difference in the direction of intermolecular NH-O-2 hydrogen bonds depending on the chirality of alanine molecule in the c(2 x 4) ordered structure. This agrees well that D- and L-alanine molecules segregate to form their own c(2 x 4) domains separated by the [110] boundary lines. (C) 2003 Elsevier Science B.V. All rights reserved.