Eosinophil cationic protein (ECP), a secretory protein of the eosinophil granulocyte, is a basic and highly heterogeneous protein. This heterogeneity is dependent on polymorphisms in the ECP gene and post-translational modifications, and it affects the functional properties of the protein in terms of cytotoxicity. The aim of this study was to further investigate the molecular heterogeneity, hence, an affinity capture assay based on an antigen-antibody interaction with the surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS) technique was developed. Of three monoclonal antibodies tested, that is, EG2, 614, and 652, the 614 mab was chosen for the experiments. ECP heterogeneity of single individuals was studied in extracts of purified blood eosinophils, and the presence of approximately 5 major molecular species was demonstrated in each subject. ECP from subjects with different ECP 434(G>C) genotypes (arg97thr) showed mass differences corresponding to the amino acid shift from arginine to threonine. ECP purified from pooled leukocytes of large numbers of healthy blood donors demonstrated an extensive mass heterogeneity with approximately 10 major molecular species. By the use of a variety of glucosidases it was shown that this heterogeneity was mainly due to N-linked oligosaccharides on which sialic acid, galactose, and acetylglucosamine was positioned. We conclude that the SELDI-TOF MS technique using specific monoclonal antibodies is a convenient and versatile tool; by means of this technique, we could detect both genetic and post-translational causes of the molecular heterogeneity of the eosinophil cationic protein.