Stimulation of insulin release by sulphonylureas is thought to involve the inhbition of K_ATP channels. The therapeutic action of sulphonylureas, however, occurs at high glucose concentrations where K_ATP channels may already be closed. This study has investigated additional ionic actions of sulphonylureas on β-cells. Islets were isolated from rat pancreas (animals killed humanely by stunning and cervical dislocation), dispersed into single cells by exposure to a low [Ca²⁺] buffer and cultured for 2-10 days. Electrical and ion channel activity were recorded using the patch-clamp technique. ⁸⁶Rb⁺ efflux (an index of net K⁺ permeability) was measured with intact perifused islets. β-cell volume was measured in isolated cells using a video-imaging technique. In the absence of glucose, tolbutamide (100 μM) caused a transient depolarisation of β-cell membrane potential. In the presence of 5 mM glucose, tolbutamide evoked a sustained period of electrical activity, whilst at 10 mM glucose the drug evoked a pronounced ‘silent’ depolarisation. Tolbutamide inhibited ^86Rb+ efflux in the absence of glucose, reflecting K_ATP channel inhibition. However, the drug caused a transient stimulation of Rb⁺ efflux in the presence of 10 mM glucose, indicative of a K_ATP channel-independent mechanism. In conventional whole-cell recordings, tolbutamide had no significant effect on activity of the volume-regulated anion channel (VRAC) current. However in intact cells, tolbutamide increased the mean amplitude (I_vrac,ave) of the VRAC current in a glucose-dependent manner with a EC₅₀ value of approximately 85 μM. For example, in the presence of 10 mM glucose, (I_vrac,ave) was −9.2 ± 0.28 pA and −16.4 ± 1.6 pA (mean ± SEM, both n=14; P<0.01 by paired t-test) in the absence and presence of 100 μM tolbutamide respectively. In single channel recordings, tolbutamide increased channel open probability from 0.11 ± 0.03 to 0.34 ± 0.08 (both n=8; P<0.01 by paired t-test) with no significant effect on current amplitude. Tolbutamide was found to increase β-cell volume by approximately 7% (P<0.001) in the presence, but not in the absence of glucose. Finally, tolbutamide-induced electrical activity in β-cells was suppressed by 4,4'-dithiocyanatostilbene-2,2'-disulphonic acid (DIDS; 100 μM and by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB; 50 μM), two VRAC inhibitors. It is concluded that tolbutamide can induce electrical activity by potentiating the VRAC current. This effect is probably not due to a direct effect of the drug on the channel, but could be secondary to an effect on β-cell glucose metabolism.