Glial cell line-derived neurotrophic factor (GDNF), a distant member of the transforming growth factor-beta superfamily, was originally purified and cloned as a potent survival factor for midbrain dopaminergic neurons. Some studies have characterized the transcriptional regulation of the GDNF gene, but its regulatory mechanisms have yet to be well defined, especially under pathophysiological conditions. In this study, we used a pharmacological approach to study the expression of the rat GDNF gene induced by lipopolysaccharide (LPS) in primary cultures of glial cells. MG132, a blocker of nuclear factor kappaB (NF-kappaB) activation, did not apparently affect LPS-induced GDNF gene expression, whereas it attenuated the up-regulation of iNOS genes via Toll-like receptor (TLR) 4. In primary glial cultures, LPS increased the phosphorylation levels of c-Jun amino-terminal kinase 1 (JNK1) and p38 mitogen-activated protein kinase (MAPK); in primary microglial cultures, it enhanced phosphorylation of extracellular signal-regulated kinase (Erk). Of the several MAP kinase inhibitors tested, a JNK-specific inhibitor blocked LPS-induced GDNF transcription in primary cultures of microglia, but not of astrocytes. These results suggest that LPS up-regulates GDNF transcription through an NF-kappaB independent pathway, and that JNK is responsible for LPS-stimulated GDNF transcription in primary cultures of microglia.