The modulation of high-threshold Ca2+ currents by the selective metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1- aminocyclopentane-1,3-dicarboxylic acid (ACPD), was investigated in cultured hippocampal neurons using whole-cell voltage-clamp recording. ACPD reduced high-threshold Ca2+ currents carried by Ba2+ with an EC50 of 15.5 microM. The inhibition was reversible, voltage dependent, and blocked by L-2-amino-3-phosphonopropionic acid (1 mM) or by pretreatment with pertussis toxin. Inhibition by ACPD was greatly enhanced, and became irreversible, when the nonhydrolyzable GTP analog GTP gamma S was included in the whole-cell pipette. In some neurons, the Ba2+ current was inhibited by L(+)-2-amino-4-phosphonobutanoic acid (L-AP4) as well as ACPD while most cells were insensitive to L-AP4, suggesting that these agonists activate distinct receptors. The inhibition of Ca2+ currents was reduced but not eliminated in the presence of either omega-conotoxin GVIA or nifedipine, suggesting that both N- and L-type Ca2+ currents were affected. The degree and kinetics of inhibition were dependent on intracellular calcium. With [Ca]i < 1 nM, inhibition had a fast onset (t approximately 1–2 sec) and a rapid recovery, consistent with a membrane-delimited pathway. However, a slow component of inhibition appeared when the steady state [Ca]i was increased to 100 nM (t onset approximately 3 min). The slow component did not require transient Ca2+ influx or release of intracellular Ca2+. We suggest that Ca2+ channel modulation by ACPD involves either two mGluR subtypes with separate coupling mechanisms or a single mGluR that couples to both mechanisms.