Colonic electroneutral NaCl absorption mechanism needs apical membrane Na⁺/H⁺ and Cl^–/HCO3^– exchangers working in parallel. The basolateral exit pathway for Na⁺ is via Na⁺/K⁺ ATPase. Recently, we have postulated that the basolateral voltage-gated chloride channel ClC-2, expressed in colonic surface epithelium, is the basolateral exit pathway for Cl^– (Catalán et al., 2004). Short chain fatty acids (SCFAs) are generated by colonic microflora and constitute the main source of energy for colonocytes. SCFAs are known to increases NaCl absorption in mammalian colon, but the molecular mechanism remains unknown. We have hypothesized that ClC-2 channel activity might be regulated by SCFAs. We have now tested whether intracellular SCFAs affect ClC-2 function. We have used the recombinant guinea-pig ClC-2 transiently expressed in HEK-293 cells and assayed by the whole-cell recording mode of the patch-clamp technique. In the presence of 35 mM intracellular Cl^– (100 mM gluconate) hyperpolarisation activated ClC-2 channel with a V_0.5 (voltage necessary to reach 50 % of maximal activation) of -108 ± 4 mV and a slope factor of -24 ± 1 mV (n=8). The reversal potential (Erev) for this current was identical to ECl (-36 mV) indicating perfect selectivity of Cl^– over gluconate. The same experiment was repeated replacing all gluconate with butyrate. Erev was not altered by butyrate replacement, indicating that butyrate does not permeate that channel. A blockade of the current by intracellular butyrate was apparent. This blockade was concentration- (IC₅₀ 25 mM) and voltage-dependent, and was rather small at physiological (>-80 mV) potentials. The activation curve was shifted to more positive potential, with a V_0.5 of -80 ± 2 mV and a slope factor -18 ± 1 mV (n=5). This effect of butyrate is similar in magnitude to that obtained after replacing gluconate with Cl^–. The effect of butyrate on V_0.5 was concentration-dependent and could be described by a Hill equation with nH of 2 and KD 22 mM. SCFAs acetate and propionate also shifted the voltage-dependence of ClC-2, but other organic acids (glutamate and lactate) did not affect ClC-2 activity. We do not know if the activity of basolateral ClC-2 channels might be important for transport of SCFAs across the basolateral membrane. Its activation by these compounds suggests that their possible involvement is worth investigating.