Single high-voltage-activated (HVA) Ca 2+ channel activity was recorded in rat insulinoma RINm5F cells using cell-attached and outside-out configurations. Single-channel recordings revealed three distinct Ca 2+ channel subtypes : one sensitive to dihydropyridines (DHPs)-(L-type), another sensitive to -conotoxin (CTx)-GVIA (N-type) and a third type insensitive to DHPs and -CTx-GVIA (non-L-, non-N-type). The L-type channel was recorded in most patches between 930 and +30 mV. The channel had pharmacological and biophysical features similar to the L-type channels described in other insulin-secreting cells (mean conductance 21 pS in control conditions and 24 pS in the presence of 5 µM Bay K 8644). The non-L-, non-N-type channel was recorded in cells chronically treated with -CTx-GVIA in the presence of nifedipine to avoid the contribution of N-and L-type channels. Channel activity was hardly detectable below 910 mV and was recruited by negative holding potentials (< 990 mV). The channel open probability increased steeply from 910 to + 40 mV. Different unitary current sublevels could be detected and the current voltage relationship was calculated from the higher amplitude level with a slope conductance of 21 pS. Channel activity lasted throughout depolarizations of 300-800 ms with little sign of inactivation. Above 0 mV the channel showed a persistent flickering kinetics with brief openings ( o 0.6 ms) and long bursts ( burst 60 ms) interrupted by short interburst intervals. The third HVA Ca 2+ channel subtype, the N-type, had biophysical properties similar to the non-L-, non-N-type and was best identified in outside-out patches by its sensitivity to -CTx-GVIA. The channel was detectable only above 910 mV from a 990 mV holding potential, exhibited a fast flickering behaviour, persisted during prolonged depolarizations and had a slope conductance of about 19 pS. The present data provide direct evidence for a slowly inactivating non-L-, non-N-type channel in insulin-secreting RINm5F cells that activates at more positive voltages than the L-type channel and indicate the possibility of identifying unequivocally single HVA Ca 2+ channels in cellattached and excised membrane patches under controlled pharmacological conditions.