Previous work from this laboratory demonstrated that arachidonic acid activates c-jun NH₂-terminal kinase (JNK) through oxidative intermediates in a Ca²⁺-independent manner (Cui X and Douglas JG. Arachidonic acid activates c-jun N-terminal kinase through NADPH oxidase in rabbit proximal tubular epithelial cells. Proc Natl Acad Sci USA 94: 3771-3776, 1997.). We now report that JNK can also be activated via a Ca²⁺-dependent mechanism by agents that increase the cytosolic Ca²⁺ concentration (Ca²⁺ ionophore A₂₃₁₈₇, Ca²⁺-ATPase inhibitor thapsigargin) or deplete intracellular Ca²⁺ stores [intracellular Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM]. The activation of JNK by BAPTA-AM occurs despite a decrease in cytosolic Ca²⁺ concentration as detected by the indicator dye fura 2, but appears to be related to Ca²⁺ metabolism, because modification of BAPTA with two methyl groups increases not only the chelation affinity for Ca²⁺, but also the potency for JNK activation. BAPTA-AM stimulates Ca²⁺ influx across the plasma membrane, and the resulting local Ca²⁺ increases are probably involved in activation of JNK because Ca²⁺ influx inhibitors (SKF-96365, nifedipine) and lowering of the free extracellular Ca²⁺ concentration with EGTA reduce the BAPTA-induced JNK activation.