This study investigates the possible involvement of serine proteases in interferon-γ (IFN-γ) activity on WISH cells. It was observed that inhibition of 3H-thymidine incorporation induced by IFN-γ was abrogated by the serine protease inhibitors Nα-tosyl-L-lysyl-chloromethane and soybean trypsin inhibitor, both of which act mainly on trypsin. Phenylmethyl sulfonyl fluoride also had a partial inhibitory effect. Other protease inhibitors specific to the cysteine, the aspartic, and the metalloprotease families were not effective. Kinetic analysis revealed that a trypsin-like protease is involved in IFN-γ activity for up to 7 h. Trypsin-like activity induced by IFN-γ was detected in the particulate fraction but not in the cytosolic fraction, whereas chymotrypsin activity was not enhanced in either the cytosolic or particulate fractions under similar conditions. Following separation on a gelatin substrate gel, two trypsin-like protease activities located in the particulate fraction were found to increase in response to IFN-γ treatment. Hence, it seems that a specific membrane-associated trypsinlike protease activity induced by IFN-γ may play a role in the action of the cytokine on thymidine incorporation in WISH cells.