An ELISA was developed using chicken cystatin as a capture agent for the immunodiagnosis of paragonimiasis and fascioliasis. The assay detected specific antibodies to fluke cysteine proteinases without the need for purified proteinases. An ELISA plate was sensitized with chicken egg white cystatin, incubated with excretory-secretory (ES) products of adult flukes, and standard ELISA procedures were then followed. The ELISA plates incubated with the ES products of Paragonimus westermani and Fasciola sp. showed high reactivity to sera from patients with paragonimiasis westermani and fascioliasis, respectively. The capture ELISA showed little cross-reactivity with sera from patients with paragonimiasis and fascioliasis, which showed immunodiagnostic cross-reactivity in a conventional ELISA using crude fluke antigens. Moreover, the capture ELISA showed little reactivity with sera from patients with five other helminth diseases and from healthy volunteers. Omitting either sensitization with cystatin or incubation with fluke ES products abolished high ELISA reactivity, as did a prior exposure of the ES products to cystatin. The addition of papain to an incubation solution of the ES products greatly reduced ELISA reactivity. Incubating the cystatin-sensitized plates with partially purified cysteine proteinases from flukes instead of the ES products also maintained a similar high ELISA reactivity. These results indicate that the cystatin capture ELISA elicits a cystatin and fluke cysteine proteinase antigen-mediated reaction and measures fluke cysteine proteinase-specific antibodies. Prior exposure to low molecular weight inhibitors of cysteine proteinases and other proteinases, such as E-64, leupeptin, aprotinin, and pepstatin, had no effect, suggesting that these inhibitors can be added to cysteine proteinase preparations to prevent autoproteolysis. This assay has good sensitivity and high specificity and is useful for the immunodiagnosis of paragonimiasis and fascioliasis.