Polyclonal antisera were prepared in rabbits using both native and chymotrypsin-digested bovine lens fiber plasma membranes. MP26, the principal protein of lens fiber plasma membranes, and CT20, a chymotryptic fragment of MP26, were isolated electrophoretically and used to purify anti-MP26 and anti-CT20 activity from the respective antisera by affinity chromatography. These affinity-purified antisera were characterized by immunoreplica. Immunofluorescence microscopy localized MP26 on sections of methacrylate-embedded lenses in the lens fiber plasma membranes, but not the lens epithelium. Immunocytochemistry of isolated native or chymotrypsin-digested lens fiber plasma membranes localized both the MP26 and the CT20 only in the nonjunctional plasma membranes, with no detectable activity in the lens fiber junctions themselves. Electron microscopy revealed a second set of pentalaminar profiles, thinner by 4 nm than the lens fiber junctions, which contained demonstrable anti-MP26 and anti-CT20 activity following immunocytochemistry. These results indicate either that MP26 is not a component of the lens fiber junctions, or that significant conformational changes accompany assembly of MP26 into lens fiber junctions, resulting in the masking of MP26 antigenic determinants.