This report presents results from experiments which evaluated the effect of exogenous protease on the in vitro antibody-forming cell (AFC) response of hamster lymphocytes to sheep erythrocytes (SRBC). In the presence of fetal calf serum, trypsin and papain, but not thermolysin, α-chymotrypsin, thrombin, and submaxillary protease, were able to enhance the quantity of AFC which developed. Prior incubation of antigen with proteases had no effect on subsequent antigenicity. The following observations were made: (1) Addition of protease to the culture system enhanced the AFC response only if added in the first 48 hr of the assay. (2) Proteases were able to enhance the development of AFC in lymph node and spleen cell cultures lacking fetal calf serum for 24 hr. (3) When papain was added to spleen cell cultures which normally produce fewer AFC than lymph node cells (LNC) it promoted the development of a 6- to 10-fold increase in AFC causing the magnitude of the response to match the AFC response expected in LNC cultures. These data support a role for a proteolytic event in lymphocyte activation by specific antigens.