Small molecules that mimic the action of cation transporters are well known. However, until recently similar molecules that transport anions have been unavailable. In previous work (Koulov et al. 2003), we demonstrated that a family of small molecules derived from cholic acid termed ‘cholapods’ bind anions with high-affinity and promote Cl^– efflux from liposomes. We have also demonstrated that cholapods induce Cl^– transport across MDCK epithelia (Koulov et al. 2003) and Cl^–-dependent currents in excised inside-out membrane patches. Because electrophysiological studies of cholapods using cells do not distinguish between (i) cholapods themselves mediating anion flow and (ii) cholapods interacting either directly or indirectly with transport proteins to induce current flow, we sought to investigate the transport activity of cholapods in an artificial environment. To achieve this goal, we modified the technique of Riquelme et al. (1990) to study cholapods in excised inside-out membrane patches from giant liposomes synthesised from the phospholipid asolectin by the dehydration/rehydration method. As a control, we studied the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channel. To measure Cl^– flow across excised inside-out membrane patches from giant liposomes, we imposed a large Cl^–-concentration gradient across the membrane patch (pipette (external) [Cl^–] = 10 mM and bath (internal) [Cl^–] = 147 mM) and clamped voltage at -50 mV. Like CFTR recorded in cellular membranes, CFTR incorporated into giant liposomes formed Cl^–-selective channels regulated by cyclic AMP-dependent phosphorylation and intracellular ATP (n = 3). We then synthesised giant liposomes with no added cell membrane to study the activity of cholapods in a similar environment. Tetrahydrofuran (THF; 0.24%, v/v), the vehicle used to solubilise cholapods caused no change in current (n = 5). In contrast, addition of the cholapod RS2 (150 μM) to the solution bathing the intracellular side of the excised membrane caused an increase in current from -0.12 + 0.04 pA to -2.43 ± 0.64 pA (n = 3-7; p < 0.05, Students t test) that was concentration dependent, but time independent (n = 4). Of note, no unitary events were discernable in the current records. In the presence of symmetrical 147 mM Cl^– solutions, the RS2-induced current had a linear current-voltage relationship with a reversible potential close to 0 mV. We interpret our data to suggest that cholapods mediate Cl^–-dependent ion transport across liposomal membranes. Our data also suggest that giant liposomes might prove a useful tool to study both incorporated ion channels and artificial transporters in isolation.