Superior cervical ganglion (SCG) sympathetic neurons die by apoptosis when deprived of nerve growth factor (NGF). It has been shown previously that the induction of apoptosis in these neurons at NGF withdrawal requires both the activity of the small GTP-binding protein Cdc42 and the activation of the c-Jun N-terminal kinase (JNK) pathway. The mixed lineage kinase 3 (MLK3) belongs to a family of mitogen-activated protein (MAP) kinase kinase kinases. MLK3 contains a Cdc42/Rac interactive-binding (CRIB) domain and activates both the JNK and the p38 MAP kinase pathways. In this study the role of MLK3 in the induction of apoptosis in sympathetic neurons has been investigated. Overexpression of an active MLK3 induces activation of the JNK pathway and apoptosis in SCG neurons. In addition, overexpression of kinase dead mutants of MLK3 blocks apoptosis as well as c-Jun phosphorylation induced by NGF deprivation. More importantly, MLK3 activity seems to increase by 5 hr after NGF withdrawal in both differentiated PC12 cells and SCG neurons. We also show that MLK3 lies downstream of Cdc42 in the neuronal death pathway. Regulation of MLK3 in neurons seems to be dependent on MLK3 activity and possibly on an additional cellular component, but not on its binding to Cdc42. These results suggest that MLK3, or a closely related kinase, is a physiological element of NGF withdrawal-induced activation of the Cdc42-c-Jun pathway and neuronal death. MLK3 therefore could be an interesting therapeutic target in a number of neurodegenerative diseases involving neuronal apoptosis.