The archaeal AUA-codon specific tRNA(Ile) contains 2-agmatinylcytidine (agm(2)C or agmatidine) at the anticodon wobble position (position 34). The formation of this essential modification is catalyzed by tRNA(Ile)-agm(2)C synthetase (TiaS) using agmatine and ATP as substrates. TiaS has a previously unknown catalytic domain, which we have named the Thr18-Cyt34 kinase domain (TCKD). Biochemical analyses of Archaeoglobus fulgidus TiaS and its mutants revealed that the TCKD first hydrolyzes ATP into AMP and pyrophosphate, then phosphorylates the C2 position of C34 with the γ-phosphate. Next, the amino group of agmatine attacks this position to release the phosphate and form agm(2)C. Notably, the TCKD also autophosphorylates the Thr18 of TiaS, which may be involved in agm(2)C formation. Thus, the unique kinase domain of TiaS catalyzes dual phosphorylation of protein and RNA substrates.