Background/Aims: The large intestine secretes HCO₃⁻ via a Cl⁻/HCO₃^post− exchange mechanism located in the apical membrane of colonocytes. However, an additional transport system(s) must facilitate HCO₃⁻ (OH⁻) entry or H⁺ exit across the basolateral cell surface. The aim of this study was to determine that mechanism(s). Methods: A modified Ussing apparatus was used to measure net HCO₃⁻ secretion in segments of rat distal colon. Results: When added to the serosal solution, 10 mmol/L 4-acetamido-4′-isothiocyano-2,2′-disulfonic acid stilbene (SITS), 1 mmol/L SITS and 0.1 mmol/L dilsothiocyanostilbene-2,2′-disulfonic acid, inhibited HCO₃⁻ secretion by 88%, 51%, and 30%, respectively. However, the Na⁺/H⁺ exchange inhibitors, amiloride (1 mmol/L), dimethylamitoride (0.1 mmol/ L), ethylisopropylamiloride (0.1 mmol/L), failed to affect HCO₃⁻ secretion. Acetazolamide (1 mmol/L) blocked HCO₃⁻ secretion by approximately 60% when in the serosal solution but had little effect when in the mucosal solution. Ion substitution studies showed that HCO₃⁻ secretion required Na⁺ in the serosal solution (K_0.5 ~ 12 mmol/L). HCO₃⁻ secretion was unaffected by depolarizing the basolateral membrane potential with K⁺-rich medium. Conclusions: These data are consistent with Na⁺ linked HCO₃⁻ transport across the colonocyte basolateral membrane, which appears to be electroneutral.