To better understand the in vivo function of secreted cysteine proteases of Haemonchus contortus, the ability of live parasites to degrade connective tissue was investigated using [3H]proline-labeled extracellular matrix produced by smooth-muscle cells (R22). The matrix was composed of glycoprotein(s) (34%), elastin (49%), and collagen (15%) in an insoluble, multilayered, cross-linked structure. No degradation of the extracellular matrix by third-stage larvae (L3) (10,000/ml) occurred during 24-hr in vitro incubation. In contrast, fourth-stage larvae (L4) (1,000/ml) degraded 42% of the matrix, whereas adults (100/ml) degraded the entire matrix. The presence of Z-phe-ala-FMK (100 microM), a specific cysteine protease inhibitor, during incubation of adults, reduced matrix degradation to 30% without affecting parasite motility. Isolated adult excretory/secretory products (ESP) (0.1 mg protein/ml) degraded 64% of the total matrix; specific degradation consisted of 80.3% of the glycoprotein, 67.1% of the elastin, and 27.6% of the collagen matrix components. Degradation of the matrix by ESP was stimulated by dithiothreitol (2 mM) and inhibited by Z-phe-ala-FMK. Thus, the secretory cysteine proteases of H. contortus are active under physiological conditions and able to degrade the major components of connective tissue in an in vitro model system that simulates their structure in vivo. These data strengthen the proposed role of these enzymes in the breakdown of host tissue.