Leishmania (Leishmania) chagasi infection activates macrophages, which release several microbicidal agents, including peptidases, to eliminate the parasite. Leishmanicidal mediators released in large amounts may cause morphological and/or functional injuries to the liver. In order to investigate the involvement of peptidases in this phenomenon, an in vitro co-culture model of peritoneal macrophages infected with L. chagasi and hepatocytes was used. High levels of released hepatic transaminases were found in supernatants from infected co-cultures at the same time point in which alterations in hepatocyte morphology and maximum proteolytic activity were observed. The largest proteolytic activity being at pH 10 as well as the greatest efficiency of treatment with 1,10-phenantroline observed in supernatants from the infected co-cultures suggests the presence of metallopeptidases during the leishmanicidal activity by infected macrophages. Furthermore, TNF-alpha levels and high levels of TGF-beta were increased at this time point, and this can be related to the synthesis of metallopeptidases and the conversion of the latent form to the active form. Metallopeptidase activities were detected by gelatin SDS-PAGE in higher amounts in infected macrophages and co-culture supernatant; moreover, one metallopeptidase migrating at 85 kDa produced in excess (41% more) by infected macrophages was identified as MMP-9. This metallopeptidase may be participating in this phenomenon together with other leishmanicidal factors released by these host cells.