Kluyveromyces lactis killer strains secrete a heterotrimeric toxin (zymocin), which causes an irreversible growth arrest of sensitive yeast cells. Despite many efforts, the target(s) of the cytotoxic γ-subunit of zymocin has remained elusive. Here we show that three tRNA species tRNA Glu mcm 5 s 2 UUC , tRNA Lys mcm 5 s 2 UUU , and tRNA Gln mcm 5 s 2 UUG are the targets of γ-toxin. The toxin inhibits growth by cleaving these tRNAs at the 3′ side of the modified wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U). Transfer RNA lacking a part of or the entire mcm 5 group is inefficiently cleaved by γ-toxin, explaining the γ-toxin resistance of the modification-deficient trm9, elp1-elp6, and kti11-kti13 mutants. The K. lactis γ-toxin is the first eukaryotic toxin shown to target tRNA.