The excretory/secretory (ES) antigen(s) of Ascaris lumbricoides was fractionated into 10 fractions by gel chromatography on a Suparose 12 column in FPLC. Of these, the third fraction (Al III), showing binding activity with both IgE and IgG antibodies of A. lumbricoides infected patients' sera, was further resolved into 2 fractions (Al IIIa and Al IIIb) on passage through a Mono Q column. Al IIIb was found to be the most potent antigen due to its high binding affinity with IgE and IgG antibodies of Ascaris infected patients as evidenced by ELISA inhibition. Although a two to five-fold increase of serum IgE level was observed in all helminthic parasite infected patients studied compared to control subjects, Al IIIb specific IgE was detected in sera of all Ascaris infected and only 40% of hookworm infected patients. When Al IIIb was tested by ELISA with sera of control subjects, Ascaris, hookworm, Strongyloides and Trichuris infected patients, strong binding was observed with the IgE and also IgG of all the Ascaris infected patients; however, it cross-reacted with IgG in 50% of hookworm, 28.6% of Trichuris trichura and 22.2% of Strongyloides infected patients' sera; but with IgE only in 40% of hookworm infected patients' sera. Further study showed specific detection of IgG4 in all the serum samples of 65 Ascaris infected patients when Al IIIb antigen was allowed to react with different subclasses of IgG by ELISA, giving a sensitivity of 100%. Reactivities of Al IIIb with IgG1 and IgG3 were only 47.6 and 11.8% respectively and there was no reactivity with IgG2 subclass. No IgG4 reactivity against Al IIIb was observed in the sera of hookworm, Trichuris or Strongyloides infected patients and was similar to that observed with control subjects showing the 100% specificity of the test system. This study may therefore be regarded as a novel technique for serodiagnosis of ascariasis by measuring Ascaris specific IgG4.