The reduction in hemozoin content is a well known feature of chloroquine-resistant Plasmodium berghei. Using NK65-derived lines displaying increasing resistance levels, we observed an inverse relationship between the hemozoin content, and the glutathione (GSH) and glutathione S-transferase (GST) levels. Treatment of highly chloroquine-resistant-infected mice with buthionine sulfoximine (BSO), which has previously been shown to partially reverse this chloroquine resistance, led to a significant increase in hemozoin production. In vitro studies on the polymerization of ferriprotoporphirin IX (FPIX) at pH 5.0 showed that GSH partially inhibited beta-hematin synthesis, while GST had a trivial and non specific effect. Furthermore, chloroquine-sensitive parasites invading reticulocytes displayed higher GSH level and GST activity, and reduced hemozoin synthesis and susceptibility to chloroquine. We conclude that, in chloroquine resistant P.berghei, GSH can detoxify hemin within the food vacuole, thus precluding its polymerization and preventing the activity of chloroquine and other quinoline-containing drugs. It is proposed that vacuolar GSH could be ascribed to an erythrocytic origin, since the resistant lines invade reticulocytes, which contain higher levels of GSH and GST than normocytes.