Previous research has established that the development and function of oligodendrocytes are influenced by glucocorticoids. The enzyme glycerol phosphate dehydrogenase (E.C.1.1.1.8) has been used as a model to study glucocorticoid regulation of gene expression in oligodendrocytes and the C6 glial cell line. In the rat brain this enzyme is exclusively localized to oligodendrocytes. The sequence of the 5Π flanking region for the rat gene encoding Glycerol Phosphate Dehydrogenase (GPDH; EC 1.1.1.8) was determined. 4 kb of sequence from the 5Π flanking region, exon 1, and part of intron 1 of the rat GPDH gene was compared to the corresponding mouse sequence. Dotplot matrix comparison revealed that the rat sequence is more than 80% similar to the mouse sequence, but differs from the mouse sequence in two regions: the rat sequence is devoid of 200 bp of B1 repeat sequence that is present in the mouse, and the rat sequence has an excess 700 bp of B2 repeat sequence inserted between Οͺ0.7 kb and Οͺ1.4 kb that is absent in the mouse. To determine the regulatory activity of the rat GPDH 5Π flanking region, various portions of the rat GPDH 5Π flanking region were placed in luciferase reporter constructs and tested for transcriptional activity. Transient transfection of reporter constructs into the C6 glial cell line revealed that the distal end of the 5Π flanking region was glucocorticoid-inducible. A 385 bp Glucocorticoid Response Unit (GRU) was identified whose glucocorticoid induction was enhanced by dibutyryl-cAMP and reduced by phorbol esters. Sequence analysis of the GRU revealed the presence of four consensus GRE sequences and other putative consensus elements. Results here suggest that the 5Π flanking region of the GPDH gene mediates the ligand-inducible regulation of GPDH, and that multiple signaling pathways converge at the 5Π regulatory sequence to modulate GPDH gene expression in oligodendrocytes.