Key features of developmentally regulated programmed cell death (PCD) have been described for the first time in the chick nervous system. JNK/c-Jun pathway was involved in early events determining normal and pathological neuronal death as shown in experimental models. In the chick embryo, PCD of motoneurons (MNs) in ovo occurs within a well-defined temporal window and can be subjected to experimental manipulation. Taking advantage of this in vivo system, we explored the role of c-Jun and JNK pathway in the regulation of PCD in MNs. By using specific antibodies against phospho-c-Jun (Ser 63, 73) and JNK we demonstrated that before MNs acquire apoptotic phenotype there is an increase in c-Jun. Blockage of neuromuscular activity by the GABA agonist muscimol reduces PCD and diminishes c-Jun immunoreactivity in MNs. Extensive induction of PCD, either due to injection of beta-bungarotoxin or limb bud removal, is also preceded by an increase in c-Jun immunoreactivity that is also associated with upregulation of phospho-c-Jun and JNK. Translocation of JNK from cytoplasm to MN nuclei was also detected. After acute application of beta-bungarotoxin, which is a strong apoptotic stimulus for MNs, c-Jun phosphorylation occurs on serine 73, whereas serine 63 is the main site for c-Jun phosphorylation after limb bud removal. These results demonstrated that the JNK/c-Jun pathway is involved in the decision phase of normal and induced apoptosis in MNs. Pharmacological interventions involving this pathway should be explored as a potential therapeutic target for promoting MN survival.