We have characterized the expression pattern and pharmacological profile of activation of metabotropic glutamate receptors (mGluRs) in immortalized, gonadotropin releasing hormone (GnRH)-secreting GT1-7 cells, which represent a homogeneous cellular population of hypothalamic origin. These cells are known to respond to the mGluR agonist (1S, 3R)-cyclopentanedicarboxylic acid (1S, 3R-ACPD) with increased GnRH release. To establish which specific mGluR subtypes are expressed by GT1-7 cells, we used polyclonal antibodies raised against non-conserved regions of the carboxy-terminal domains of individual subtypes. The selectivity of these antibodies was tested in HEK 293 cells transiently transfected with each mGluR subtype. GTl-7 cells stained positively for the subtypes mGluRla, -1b and -5 (belonging to group I mGluRs), mGluR2/3 (group 11) and mGluR7 (group 111). Agonists of group I mGluRs, including 1S, 3R-ACPD, activated phosphoinositide hydrolysis in GT1-7 cells. This effect, however, was manifested only when cell density was low, and it disappeared when cells reached confluence. Stimulation of phosphoinositide hydrolysis could not therefore have been related to hormone secretion because 1S, 3RACPD effectively released GnRH in confluent cultures. We then focused on group II and Ill mGluRs, which in transfected cells are negatively linked to adenylate cyclase activity. Unexpectedly, however, agonists which preferentially activate group II and Ill mGluRs increased both basal and forskolin-stimulated cAMP accumulation in GT1-7 cells. Stimulation of GAMP accumulation by mGluR agonists was not prevented by enzymatic depletion of endogenous adenosine, but was obliterated when cells were incubated with agonists of receptors positively coupled to adenylate cyclase, such as P-adrenergic and prostaglandin E2 receptors. These results suggest that GT1-7 cells express a novel mGluR subtype positively coupled to adenylate cyclase, which shares the same transduction pathway of other classical receptors coupled with a G,-type of GTP-binding protein.