The mode of inhibition of N-methylisatin-β-4',4'-diethylthiosemicarbazone (M-IBDET) on Moloney leukemia virus production was studied. Drug treatment of infected cells did not alter the amounts or sizes of the 35S and 22S subgenomic viral RNAs. The translation abilities of poly(A)+ RNA derived from M-IBDET-treated cells was also unaffected, as judged by cell-free translation analysis. Poly(A)+ RNA derived from M-IBDET-treated cells directed translation of equal amounts of viral gag precursors, gPr-80(gag) and Pr-65(gag), as did poly(A)+ RNA prepared from untreated cells. The addition of M-IBDET to a cell-free translation system programmed with either total poly(A)+ RNA extracted from infected cells or hybrid-selected viral RNA inhibited the synthesis of viral protein precursors. An examination of the effect of M-IBDET on polysomes engaged in the translation of viral proteins revealed a fourfold accumulation of polysomal virus-specific RNA in drug-treated cells. These results suggest that the inhibition of Moloney leukemia virus by M-IBDET involves a block in the translation of viral RNA rather than interference with viral RNA transcription.