Irradiation has been successful in the attenuation of infective stage parasites for use as vaccines against a number of parasites including Fasciola spp. The mechanisms of action of irradiation-attenuated vaccines, however, are not clearly understood. In this study, we examined the effect of 3, 10, and 40 krad of gamma-irradiation on the expression of carbohydrates and cathepsin-B by newly excysted juvenile Fasciola hepatica (NEJ). Following irradiation of metacercariae, the expression of concanavalin A (ConA)-specific sugars was decreased on the surface of NEJ and the expression of wheat germ agglutinin (WGA)-specific sugars was increased in the gut and reduced on the surface of NEJ. Cathepsin proteases are a major component of liver fluke excretory/secretory material (ES) and can cleave host immunoglobulin (Ig). Cathepsin-B protease was localized in nonirradiated NEJ to the gut lumen and to secretory granules within the gut epithelia. Irradiation of fluke with 3, 10, and 40 krad of gamma-rays significantly reduced the tissue expression of cathepsin-B at 8 hr postirradiation in an apparently dose-dependent manner. After a further 24 hr culture tissue expression of cathepsin-B was significantly reduced in 10- and 40-krad-irradiated NEJ. Protease activity of ES samples collected over a 24-hr period from irradiated and nonirradiated NEJ cultured in vitro were tested using a rabbit Ig cleavage assay. The proteolytic activity of ES from 10- and 40-krad-irradiated NEJ was reduced during the initial 6 hr in culture and between 12 and 24 hr when compared to ES from nonirradiated controls. Biosynthetic labeling experiments using [35S]methionine and [35S]cysteine indicated that ES material was actively synthesised during 48 hr in vitro culture. Therefore, from this study, we conclude that gamma-irradiation of NEJ alters expression of cathepsin-B protease and WGA- and ConA-specific sugars which may be detrimental to parasite invasion and contribute to the protective immune responses generated in the host by irradiation-attenuated metacercariae of Fasciola spp.