The trichostrongylid nematode Haemonchus contortus released a hyaluronic acid-degrading enzyme during in vitro development from the third (L3) to fourth (L4) larval stage. The enzyme did not degrade chondroitin sulfate A. Enzyme activity was optimal between pH 4.0 and 6.0, and the enzyme was inhibited by high concentrations of NaCl; the divalent cations Cu2+, Zn2+, Ca2+, and Mn2+ were not inhibitory. The hyaluronidase had a molecular mass estimated at 57 kDa by sucrose density gradient centrifugation and at 111 kDa by substrate sodium dodecyl sulfate polyacrylamide gel electrophoresis (reducing and nonreducing conditions), suggesting the formation of a dimer during the electrophoretic separation conditions. The level of hyaluronidase released during in vitro development peaked between 24 and 48 hr in culture and then gradually decreased, with little or no activity present in the 168-hr culture fluid. The enzyme was not detected in culture fluid from 24-hr incubations of either the mid-L4 stage (obtained from sheep 7 days postinfection) or the adult stage (obtained from sheep 30-35 days postinfection). The temporal expression of the hyaluronidase suggested a role for this enzyme in the early stages of the L3-L4 developmental process.