Surface iodinated larvae of Haemonchus contortus were incubated in the presence of the fungal protease, proteinase K, and proteolysis quantified by scintillation counting of released radioactivity. No radioactivity was released from live ensheathed infective-stage larvae (L3(2M)). In contrast, 58% of the radioactivity was released from ecdysed, second molt (2M) cuticles (sheaths) of L3(2M) and 48% from live exsheathed third-stage larvae (L3). When L3(2M) larvae were killed by heat (80 degrees C for 10 min) prior to proteinase K incubation, 61% of the radioactivity was released, whereas less than 7% was released from larvae killed by the metabolic inhibitors NaN3 or KCN. Proteinase K released 44% of the radioactivity from live L3(2M) larvae which had been preincubated with 1% sodium dodecylsulfate (SDS), whereas no radioactivity was released from L3(2M) larvae preincubated with either 1% Triton X-100, 0.2% CTAB, 50% methanol, 50% ethanol, or water. Following incubation with proteinase K, only L3(2M) larvae which had been heat-killed or preincubated with SDS showed visible damage to the sheath. Material released from L3(2M) larvae by exposure to either heat or SDS contained a 98,000 M(r) protein by SDS-PAGE autoradiography. These results indicate that viable L3(2M) larvae are resistant to attack by proteinase K and that this resistance is dependent on structural properties of the sheath.