The transcriptional regulation of the murine gene encoding the 67-kDa form of glutamic acid decarboxylase (GAD67) was studied by β-galactosidase histochemistry in transgenic mice carrying fusion genes between progressively longer portions of the 5′-upstream regulatory region of GAD67 and E. coli lacZ. No expression was detected in brains of mice carrying 1.3 kb of upstream sequences including a housekeeping and two conventional promoters, and two negative regulatory elements with homology to known silencers. In mice carrying the same portion of the promoter region plus the first intron, lacZ expression in the adult central nervous system was found in few, exclusively neuronal sites. The number of correctly stained GABAergic centres increased dramatically with increasing the length of the 5′-upstream region included in the construct which suggests that multiple putative spatial enhancers are located in this region. Their action is influenced by epigenetic mechanisms that may be due to site-of-integration and transgene copy-number effects. Additional cis-acting elements are needed to obtain fully correct expression in all GABAergic neurons of the adult central nervous system.