The mechanism through which cisplatin (cis-diamminedichloroplatinum) inhibits protein synthesis in rabbit reticulocyte lysate was characterized. Cisplatin and transplatin caused a progressive slowing in the rate of protein synthesis culminating in the complete arrest of translation. Inhibition was dependent upon the aquation of the compounds. Addition of eukaryotic initiation factor eIF-2, eIF-2B, cAMP, MgGTP, or dithiothreitol neither prevented nor reversed the inhibition induced by cisplatin, indicating that the mechanism of cisplatin-induced translational inhibition is distinct from the inhibition induced by other toxic heavy metal ions (Hurst, R., Schatz, J. R., and Matts, R. L. (1987) J. Biol. Chem. 262, 15939-15945; Matts, R. L., Schatz, J. R., Hurst, R., and Kagen, R. (1991) J. Biol. Chem. 266, 12695-12702). Analysis of the polyribosome profile of cisplatin-inhibited reticulocyte lysate indicated that cisplatin arrests the elongation stage of protein synthesis. Agarose gel electrophoresis and Northern blot analysis indicated that mRNA and rRNA become crosslinked to form very high-molecular-weight adducts upon extraction of the RNA from polyribosomes of cisplatin-treated lysates. Diethyldithiocarbamate, which reduces the cytotoxicity of cisplatin in vivo, protects protein synthesis in reticulocyte lysate from inhibition by cisplatin. The data suggest that extensive derivatization of reticulocyte lysate RNA by cis- and transplatin results in the arrest of translating ribosomes. Since arrest of translational elongation is a well-defined mechanism of action of several families of toxins, we suggest that it may contribute to the cytotoxic action of cisplatin observed in certain populations of cells.