The effect of glutamate on the accumulation of [3H]inositol phosphates was examined in oligodendrocyte progenitor cultures prepared from rat brains. Glutamate, and the analogues alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate, caused a concentration- and time-dependent increase in [3H]inositol trisphosphate (IP3) formation and the effect was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a competitive AMPA and kainate receptor antagonist. Similarly, the more selective, noncompetitive antagonist of AMPA receptors, 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466), significantly reduced the effect of both AMPA and kainate. In contrast, antagonists of N-methyl-D-aspartate (NMDA) receptor, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclo-hepten-5, 10-imine (MK-801) and R(-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), and antagonists of metabotropic receptors, L(+)-2-amino-3-phosphono-propanoic acid (L-AP3) and alpha-methyl-4-carboxyphenylglycine (MCPG), were ineffective. These results suggest that the effect of glutamate on [3H]IP3 accumulation is mediated through ionotropic AMPA receptors. Cyclothiazide, an inhibitor of AMPA receptor desensitization, strongly potentiated the AMPA and kainate-stimulated [3H]IP3 formation as well as the uptake of 45Ca2+ in line with the previous findings. 45Ca2+ uptake evoked by AMPA or kainate, in combination with cyclothiazide, was also prevented by both CNQX and GYKI 52466. Glutamate-stimulated [3H]IP3 accumulation was prevented by EGTA, suggesting a requirement for extracellular calcium. Pre-incubation with the voltage-gated Ca2+ channel blockers, diltiazem, nifedipine and CdCl2, partially prevented the glutamate-induced [3H]IP3 accumulation as well as 45Ca2+ uptake. Similarly, the Na+/Ca2+ exchanger blockers benzamil and 3,4-dichlorobenzamil reduced significantly kainate-stimulated 45Ca2+ uptake. These data indicate that glutamate-induced [3H]IP3 accumulation is triggered by calcium influx via AMPA receptors, voltage-gated calcium channels and the Na+/Ca2+ exchanger operating in reverse mode.