We have studied the biochemical and functional aspects of omega-conotoxin GVIA (omega-CgTx)-sensitive calcium channels in cerebellar granule cells in vitro. 125I-omega-Conotoxin GVIA (125I-omega-CgTx) binding sites were detected in intact cultured cerebellar granule cells and binding parameters were measured (Bmax: 134 fmol/mg protein; kinetic association constant kappa: 3.10(6) M-1.s-1). [3H]Glutamate release was assessed under different release paradigms (namely release triggered by calcium, voltage, and sodium channel agonists) and different times (15 s and 2 min). However, in all cases, [3H]glutamate release was found to be completely insensitive to omega-CgTx. Conversely, voltage-dependent release was inhibited in a dose-dependent fashion by cadmium chloride, with total inhibition at 10(-4) M. These results indicate that N-type calcium channels are not involved in glutamate secretion from granule neurons.