We have developed a modified assay method for the measurement of antibodies against PT and FHA. Polystyrene balls coated with purified PT and FHA were incubated with standard or test sera, washed, incubated with goat anti-human IgG F(ab')2 conjugated with horseradish peroxidase and then washed again. Peroxidase activity was then measured by color development using o-phenylenediamine. The anti-PT and anti-FHA IgG titers were determined using calibration curves. The assay standard curves for anti-PT and anti-FHA were both linear from 1 to 100 ELISA units per milliliter (EU ml-1), and the assay sensitivity was calculated to be 1 EU ml-1 in both cases. Intra- and inter-assay CVs were both less than 10%. Inter-laboratory assay CVs calculated from the results from four different facilities were 3.3-34.6% (average 14.5%) and 2.1-30.3% (average 13.6%) for anti-PT and anti-FHA IgG, respectively, and the correlation coefficient between the Chinese hamster ovary (CHO) cell assay and the anti-PT PS-ball ELISA assay was 0.959. Using this assay method we obtained in a highly sensitive and reproducible measurement of IgG antibodies against PT and FHA in human sera. This assay should be especially useful for the determination of low concentrations of these antibodies.