In primary cultures of mouse cerebellar granule cells, a brief stimulation by gamma-hydroxybutyric acid (GHB, 0.1-3 mM) significantly increased the intracellular Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner. In addition, gel mobility assay showed that exposure of the cells to GHB also increased nuclear DNA-binding activity specific for the cyclic AMP-responsive element (CRE) and activator protein 1 (AP-1) transcriptional element in a concentration-dependent manner. The concentration range of GHB that increased the DNA-binding activity was essentially the same as the concentration range that elicited the increase in [Ca2+]i. The GHB-induced increases in [Ca2+]i and nuclear DNA-binding activity were antagonized by specific GABAB antagonists such as p-[3-aminopropyl]-p-diethoxymethylphosphinic acid (CGP 35 348) and 3-N-[1-(S)-(3,4-dichlorobenzyl)ethanol-2-(S)-hydroxy-P-benzylphosphin ic acid (CGP 55 845). In addition, the GHB-induced increase in [Ca2+]i was abolished by pretreatment of the cells with islet-activating protein. Furthermore, treatment of the cells with 1,2-bis(2'-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM) and thapsigargin blocked the GHB-induced increase in nuclear DNA-binding activity. GHB inhibited [3H]baclofen binding to cultured cerebellar granule cells and mouse cerebellar membranes. These results suggest that stimulation of GABAB receptors by GHB activates intracellular Ca2+ stores and that the increased [Ca2+]i resulting from release of stored Ca2+ plays an important role in increasing the CRE- and AP-1 DNA-binding activities in cultured cerebellar granule cells.