A new, practical method for determining antibody to pertussis toxin (PT) by enzyme-linked immunosorbent assay for public health and clinical laboratories is possible because of recent advances in understanding the pathobiology of Bordetella pertussis and the physicochemical properties of PT. The new approach does not require the use of highly purified PT antigen, which is difficult and expensive for most laboratories to obtain. Moreover, it employs only reagents that are commercially readily available. The method combines the purification of PT antigen and an assay for PT antibody into one process. It depends on (i) growth of B. pertussis in a simple, defined medium to obtain a PT-rich supernatant with little contamination of cellular antigens; (ii) a simple, one-step concentration of PT in the culture supernatant with Affi-Gel Blue (Bio-Rad Laboratories, Richmond, Calif.); and (iii) specific adsorption of PT as the test antigen to microtiter wells coated with fetuin for the enzyme-linked immunosorbent assay. The procedure is sensitive and specific for PT antibody. It is technically simple, reproducible, and can be performed in a modestly equipped laboratory.