The widespread prokaryotic toxin-antitoxin (TA) systems involve conditional interaction between two TA proteins. The interaction between the Epsilon and Zeta proteins, constituting the TA system of plasmid pSM19035 from Streptococcus pyogenes, was detected in vivo using a yeast two-hybrid system. As we showed using Saccharomyces cerevisiae, the Zeta toxin hybrid gene also exerts its toxic effects in a dose-dependent manner in eukaryotic cells. Analysis of mutant proteins in the two-hybrid system demonstrated that the N-terminal part of Zeta and the N-terminal region of Epsilon are involved in the interaction. The N-terminal region of the Zeta protein and its ATP/GTP binding motif were found to be responsible for the toxicity.