c-Jun N-terminal kinase (JNK) is a stress signal transducer linked to cell death, and survival. JNK1 has been implicated in obesity, glucose intolerance, and insulin resistance. In this study we report the kinetic mechanism for JNK1β1 with transcription factors ATF2 and c-Jun along with interaction kinetics for these substrates. JNK1β1 followed a random sequential mechanism forming a ternary complex between JNK-substrate-ATP. K(m) for ATF2 and c-Jun was 1.1 and 2.8 μM, respectively. Inhibition studies using adenosine 5'-(β,γ-methylenetriphosphate) and a peptide derived from JNK interacting protein 1 (JIP1) supported the proposed kinetic mechanism. Biolayer interferometry studies showed that unphosphorylated JNK1β1 bound to ATF2 with similar affinity as it did to c-Jun (K(D) = 2.60 ± 0.34 versus 1.00 ± 0.35 μM, respectively). The presence of ATP increased the affinity of unphosphorylated JNK1β1 for ATF2 and c-Jun, to 0.80 ± 0.04 versus 0.65 ± 0.07 μM, respectively. Phosphorylation of JNK1β1 decreased the affinity of the kinase for ATF2 to 11.0 ± 1.1 μM and for c-Jun to 17.0 ± 7.5 μM in the absence of ATP. The presence of ATP caused a shift in the K(D) of the active kinase for ATF2 to 1.70 ± 0.25 μM and for c-Jun of 3.50 ± 0.95 μM. These results are the first kinetic and biochemical characterization of JNK1β1 and uncover some of the differences in the enzymatic activity of JNK1β1 compared with other variants and suggest that ATP binding or JNK phosphorylation could induce changes in the interactions with substrates, activators, and regulatory proteins.