The present study investigated the segmental discrepancy of the rat distal colonic anion transport induced by luminal forskolin and the possible underlying mechanisms using short-circuit current recording technique and comparative quantity real-time PCR analysis. Forskolin-induced I_SC in the segment next to lymph node (DC₁) and the segment 4 cm away from lymph node (DC₄) were 4.09±0.66 μA/cm² and 18.84±3.18 μA/cm² (n=13), respectively, which were blocked by luminal Cl⁻ channel blocker, glybenclamide (1 mM) (n=5, p<0.01), as well as removal of extracellular Cl⁻ and HCO₃⁻ in both DC₁ and DC₄ (n=5, p<0.001). Furthermore luminal pretreatment with K⁺ blockers, TEA (5 mM) and glybenclamide (100 μM) didn't affect forskolin and bumetanide-enhanced I_SC. Reducing serosal Cl⁻ concentration increased forskolin-induced I_SC by 90% in DC₁ but decreased forskolin-induced I_SC in DC₄ by 50%. Furthermore, pretreatment with serosal bumetanide, an inhibitor of Na⁺-K⁺-2Cl⁻ cotransporter, enhanced forskolin-induced I_SC by 87% in DC₁, from 4.09±0.66 μA/cm² to 7.65±0.53 μA/cm² (n=6, p<0.01), but inhibited forskolin-induced I_SC by 50% in DC₄, from 29.19±4.51 μA/cm² to 15.06±4.10 μA/cm² (n=6, p<0.05). Pretreatment with luminal amiloride (10 μM), an inhibitor of ENaC, and serosal 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) (200 μM), an inhibitor of NBC, significantly inhibited the forskolin-induced I_SC in DC₁ (n=6, p<0.05), but not in DC₄. Real-time PCR analysis did not show the significant differences between the two segments in the expression amounts of CFTR and NKCC mRNAs, however the expression of NBC mRNA in DC₄ was significantly higher than that in DC₁. In conclusion, the rat distal colon manifests a segmental discrepancy in anion transport stimulated by luminal forskolin. HCO₃⁻ might be predominantly involved in the forskolin-induced anion secretion in DC₁, but Cl⁻ might be the main component of the anion secretion in DC₄.