The mechanisms which permit Leishmania to survive inside macrophages are not totally understood although it is known that prolonged culture in vitro results in loss of virulence. One of the cell surface molecules often implicated in virulence mechanisms is the glycoprotein of 63 kDa (gp63). In this work we studied changes in infectivity of L. infantum promastigotes maintained in vitro by subcultures, correlated with the proteolytic activity of gp63. It was observed that L. infantum MON-1 promastigotes became unable to establish an infection after 6 subcultures in vitro independently of the size of inoculum. This corresponded to a diminution of proteolytic activity of gp63. L infantum MON-1 promastigotes inoculated in hamsters viscera-lize in the mononuclear phagocytic system accompanied by an antibody response. A correlation between antibody response, inoculum size and promasti gote origin was verified. L donovani MON-18 and L. infantum MON-24 promastigotes produced a specific humoral response but failed to establish an infection in hamsters regardless of all the passages tested.