Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel is inhibited by a broad range of intracellular organic anions. Here it is shown, using patch clamp recording from CFTR-transfected mammalian cell lines, that the fatty acids butyrate and 4-phenylbutyrate cause a voltage-dependent block of CFTR Cl⁻ currents when applied to the cytoplasmic face of membrane patches, with apparent K_ds (at 0 mV) of 29.6 mM for butyrate and 6.6 mM for 4-phenylbutyrate. At the single channel level, both these fatty acids caused an apparent reduction in CFTR current amplitude, suggesting a kinetically fast blocking mechanism. The concentration-dependence of block suggests that CFTR-mediated Cl⁻ currents in vivo may be affected by both 4-phenylbutyrate used in the treatment of various diseases, including cystic fibrosis, and by butyrate produced endogenously within the colonic lumen.