We previously reported that a small peptide based on amino acids 143-153 of the c-Jun N-terminal kinase (JNK)-binding domain of JIP-1 functioned as an in vitro inhibitor of JNK activity. This peptide (TI-JIP: RP-KRPTTLNLF) resembles the kinase-interaction motif (KIM = (K/R)(2-3)X1-6(L/I)X(L/I)), which is common to upstream activators, downstream substrates, phosphatases, and scaffold proteins present in MAPK cascades. In this study, we characterized the mechanism of JNK inhibition by this peptide and further investigated the biochemical features of this peptide resulting in potent JNK inhibition. We also tested various KIM-based peptides for their ability to inhibit JNK activity. TI-JIP was found to be competitive with respect to the phosphoacceptor substrate c-Jun (K-I = 0.39 +/- 0.08 muM), and exhibit mixed (non-competitive) inhibition with respect to ATP. All seven substitutions of Pro-5 we tested significantly reduced the JNK inhibition, as did altering the Pro-5 to Leu-8 spacing. When we independently tested eight substitutions of either Thr-6 or Thr-7, only one substitution in each position was well tolerated. Furthermore, peptides based on the KIMs from other proteins were significantly less potent JNK inhibitors than TI-JIP, including a peptide from the JNK interactor Sab that contained all critical inhibitory residues present in TI-JIP. Therefore, despite having previously identified Arg-4, Pro-5, Leu-8, and Leu-10 in TI-JIP as independently critical for mediating JNK inhibition, we find their presence in other 11-mer peptides is not sufficient for JNK inhibition. TI-JIP is therefore a unique KIM-based inhibitor of JNK activity.