Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) bind to GFRα-1 and GFRα-2 receptors, respectively, and their neurotrophic activity is mediated by the tyrosine kinase receptor, Ret. All these molecules were found to be expressed in primary cultures of rat glial cells, which were largely composed of astrocytes and maintained in serum-free medium. Although GDNF, NTN and Ret mRNA levels were at the limit of detection, RNase protection assays revealed relatively high amounts of GFRα-1 and GFRα transcripts. To characterize signals controlling their expression, glial cells were exposed to serum or treated with hormones acting through nuclear receptors and by activators of the cAMP or protein kinase C (PKC)-dependent pathways. Retinoic acid or 1,25-dihydroxyvitamin D3 appeared ineffective. In contrast, the 5-fold increase in GFRα-2 mRNA after 24 hr of treatment with 10−10 M of tri-iodothyronine, suggests a physiological role of thyroid hormone in the regulation of this receptor in vivo. The serum induced a 7-fold increase in GFRα-1 mRNA levels. These changes may be mediated by the cAMP or PKC pathways because both forskolin and TPA up-regulated the GFRα-1 gene. Interestingly, only TPA led to a coordinated increase in the levels of GDNF, GFRα-1 and GFRα-2 mRNAs. On the other hand, NTN transcripts remained constant, irrespective of the culture conditions. Taken together, these results indicate that GDNF family ligands and their receptors are regulated in glial cells by common or independent transductional pathways, which could modulate their specific expression during brain development or in the case of trauma. J. Neurosci. Res. 64:242–251, 2001. © 2001 Wiley-Liss, Inc.