The glial cell line-derived neurotrophic factor (GDNF) has been proven to be the most powerful neurotrophic factor in neuronal development. However, it remains uncertain as to which intracellular signaling pathway interacting with GDNF is invovlved in motoneuron (MN) development. In this study, we investigated whether phosphoinositide phospholipase C-γ (PLC-γ) is involved in GDNF-promoted MN development. The primary spinal MNs from 12- to 14-day-old embryos of Sprague-Dawley rats were cultured and survival was sustained by GDNF. A specific inhibitor of PLC-γ, 1-[6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl) amino)hexyl]-1H-pyrrole-2,5-dione (U73122), was used to block the pro-survival effect of GDNF. Our results showed that MN-like cells appeared at 72 h after initial implantation and were sustained for a period of up to seven days under GDNF treatment. These cultured MNs expressed neuron-specific enolase, SMI-32, 75-kDa low-affinity neurotrophic receptor and choline acetyltransferase. The survival rate of the cultured MNs at 24 h was significantly lower in the GDNF + U73122-treated group (31.87±2.17%), compared either with that of the GDNF- (81.38±1.13%) or GDNF + DMSO (79.39±1.22%)-treated groups. The present data suggest that PLC-γ may be one of the intracellular signals that play a role in the survival-promoting effects of GDNF in developing spinal MNs.