Fish which survive Ichthyophthirius multifiliis infection become resistant to subsequent challenge, and their sera immobilize the parasite in vitro. We have postulated that immobilizing antibodies play a role in this acquired protective immunity. Murine monoclonal antibodies (mAbs) with the ability to immobilize the parasite in vitro were produced in order to identify and purify antigens involved in immobilization. Two membrane glycoprotein immobilization antigens (i-ags) with approximate molecular masses of 43 kD and 52 kD were copurified by mAb affinity chromatography. Channel catfish fingerlings injected intraperitoneally (ip) with immobilizing mAbs against these antigens were completely protected from infection when challenged with the parasite 24 hours later. In two replicate vaccination trials, fish injected ip with membrane protein fractions enriched with the 43 kD and 52 kD i-ags had a mean survival rate of 44.7% (S.D.+17.3%) following challenge with a lethal number of parasites. These results support the hypothesis that I. multifiliis i-ags are involved in eliciting a protective immune response against the parasite.