The whole-cell and perforated patch configurations of the patch-clamp technique were used to characterise the volume-sensitive anion channel in rat pancreatic β-cells. The channel showed high permeability (P ) relative to Cl- to extracellular monovalent organic anions (PSCN/PCll = 1.73, Pacetate/PCll = 0.39, Plactate/PCll = 0.38, Pacetoacetate/PCll = 0.32, Pglutamate/PCll = 0.28) but was less permeable to the divalent anion malate (Pmalate/PCll = 0.14). Channel activity was inhibited by a number of putative anion channel inhibitors, including extracellular ATP (10 mM), 1,9-dideoxyforskolin (100 µM) and 4-OH tamoxifen (10 µM). Inclusion of the catalytic subunit of protein kinase A in the pipette solution did not activate the volume-sensitive anion channel in non-swollen cells. Furthermore, addition of 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) or forskolin failed to activate the channel in intact cells under perforated patch conditions. Addition of phorbol 12,13-dibutyrate (200 nM), either before or after cell swelling, also failed to affect channel activation. Our findings do not support the suggestion that the volume-sensitive anion channel in pancreatic β-cells can be activated by protein kinase A. Furthermore, the β-cell channel does not appear to be subject to regulation via protein kinase C. Experimental Physiology (2001) 86.2, 145-150.