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Jasmonic acid (JA) and its derivatives are involved in mobile signaling in plants during defense against biotic and abiotic stress. In our recent paper, it was found that exogenously applied jasmonoyl isoleucine (JA-Ile) in biological concentration is transported to distal sites in wounded tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum). Here we investigated the distal transport of de novo-synthesized JA-Ile in response to leaf wounding in tobacco and tomato using [^2H_1-10, ^2H_2-11, ^2H_3-12] JA ([^2H_6]JA) and [U-^<13>C]L-Ile ([^<13>C_6]Ile). [^2H_6]JA-[^<13>C_6]Ile, [^2H_6]JA-Ile, and JA-[^<13>C_6]Ile were recovered in distal untreated leaves of tobacco and tomato after treating wounded leaves with [^2H_6]JA together with [^<13>C_6]Ile. JA-Ile applied exogenously to wounded tissues reached distal tobacco and tomato leaves within 10 min, and wound-induced accumulation of JASMONATE ZIM3 (JAZ3) mRNA in distal undamaged tomato leaves was observed within 2 h. In distal undamaged leaves of the tomato spr2 mutant, which has abnormal JA and JA-Ile synthesis, JAZ3 and PROTEASE INHIBITOR II (PIN II) mRNA accumulation were induced by applying [^2H_6]JA-Ile to wounded leaves. These results suggest that JA-Ile acts as a mobile signal involved in the systemic wound response against biotic and abiotic stress.