Among the various biochemical functions assumed by the tripeptide glutathione (GSH), a role in cell protection against xenobiotics has been well established. In the case of resistance to cis-diamminedichloroplatinum(II) (CDDP) this role is controversial. CDDP reacts with nucleophiles and binds covalently to DNA, its ultimate target. We addressed the question of a putative role of GSH as a secondary non-essential target by using a bacterial model. With an Escherichia coli K12 mutant devoid of GSH, we found sensitivity to CDDP increased by a factor of two. It appeared that GSH protects bacteria at least by covalently trapping platinum before its binding to DNA since (i) lower binding of CDDP to DNA was found when GSH was present and (ii) the resistance still persisted in bacteria after treatment by the monofunctional derivative [Pt(dien)Cl]Cl. On the other hand, with a DNA repair defective mutant (lexA3), we found that other biochemical secondary target(s) might be involved in bacterial protection at low CDDP concentrations.