CLICs are ubiquitous but controversial ion channel proteins that often coexist in soluble and integral membrane forms (1). Although their roles remain unclear, the C. elegans CLIC-like protein exc-4 appears to be essential for intracellular vesicle fusion (2), supporting the idea that CLICs contribute to charge-compensating intracellular anion channels. Soluble CLICs are structurally similar to Ω-type glutathione-S-transferases (3), but the structure of the membrane proteins remains elusive. Soluble, recombinant, human CLIC1 and rat brain CLIC4 (p64H1) were incorporated into planar lipid bilayers. CLIC1 formed channels of 38 ± 3 pS (mean ± SD, n = 23) under strongly-reducing conditions with 500 mM KCl on the ‘cytosolic’ side and 50 mM KCl on the opposite (‘luminal’) side, while CLIC4 formed channels of 10.3 ± 1.0 pS (mean ± SD, n = 15). CLIC1 was mildly anion selective (Pa/Pc = 1.4 ± 0.25 n = 23), whereas CLIC4 (despite its name) was mildly cation-selective (Pa/Pc (relative permeability of anions vs cations) = 0.54 ± 0.09, n = 15, P <0.01 by t test). In glutathione buffers both channels showed a 'smooth' reduction in amplitude on oxidation, previously attributed (in CLIC1) to unstable disulphide bond formation between pairs of membrane subunits (4). We identified a putative pore-forming region based on hydropathy plots, protease digestion studies, the sidedness of the N- and C-termini, and exc-4 truncation studies, and tested the idea that CLIC channels contain at least 4 subunits each with a single transmembrane domain (TMD). In this simple model, the luminal (or external) side of each subunit contains a single cysteine residue located just before the putative pore entrance. Consistent with these ideas, truncated proteins comprising the first 58 residues of CLIC1, or the first 61 residues of CLIC4 (sufficient in each case to contain the putative TMD), autoinserted into bilayers to form redox-sensitive ion channels. 0.2 mM thiol-reactive dithiobis-nitrobenzoic acid (DTNB) blocked both the full-length and truncated channels, but only from the luminal side. The truncated proteins showed a reduced conductance, and were non-selective between anions and cations. The very poor ionic selectivity of full-length 'CLIC' channels may be conferred by the channel vestibules rather than the (very similar) residues lining the putative pore, and charged residues in the vestibule may 'concentrate' permeant ions and increase the single-channel conductance (cf (5)).