We have established a series of monoclonal antibodies that bind to phosphatidylserine (PS). One mAb, PS4A7, showed a strict specificity for PS and distinguished the stereospecific configuration of its serine moiety. We determined the amino acid sequences of the heavy and light chain variable regions of PS4A7, and examined the reactivity of the synthetic peptides corresponding to the complementarity determining region (CDR) of the mAb with phospholipids. We found that a 12-amino acid synthetic peptide corresponding to the third CDR of the heavy chain (amino acid residues 93-102, referred to as CDR3-H) bound specifically to PS. Although the affinity of the peptide to PS was markedly lower, the peptide was shown to bind to 1,2-diacyl-sn-glycero-3-phospho-L-serine (PS), but not to 1,2-diacyl-sn-glycero-3-phospho-D-serine, showing a similar specificity to that of PS4A7. The specific binding of the CDR3-H peptide to PS was confirmed by ELISA and TLC-immunostaining assay. The interaction between the CDR3-H peptide and water-soluble PS-derivatives was investigated by inhibition of the ELISA. PS effectively inhibited the binding and phosphoserine showed a weak but significant inhibition, but no appreciable inhibition was observed with serine. These observations suggest that the CDR3-H peptide plays a major role in the interaction of PS4A7 with the phosphoserine residue of the PS molecule.