Basolateral Na⁺HCO₃^- cotransport is essential for intestinal anion secretion, and indirect evidence suggests that it may be stimulated by a rise of intracellular cAMP. We therefore investigated the expression, activity and regulation by cAMP of the Na⁺HCO₃^- cotransporter isoforms NBC1 and NBCn1 in isolated murine colonic crypts. Na⁺HCO₃^- transport rates were measured fluorometrically in 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF)-loaded crypts, and mRNA expression levels and localization determined by semiquantitative PCR and in situ hybridization. Acid-activated Na⁺HCO₃^- cotransport rates were 5.07±0.7 mM/min and increased by 62% after forskolin stimulation. NBC1 mRNA was more abundant in colonic crypts than in surface cells, and crypts expressed far more NBC1 than NBCn1. To investigate whether the cAMP-induced Na⁺HCO₃^- cotransport activation was secondary to secretion-associated changes in HCO₃^- or cell volume, we measured potential forskolin-induced changes in pH_i, and assessed Na⁺HCO₃^- transport activity in CFTR -/- crypts (in which no forskolin-induced cell shrinkage occurs). We found 30% reduced Na⁺HCO₃^- transport rates in CFTR -/- compared to CFTR +/+ crypts, but similar Na⁺HCO₃^- cotransport activation by forskolin. These studies establish the existence of a [HCO₃^-]_i- and cell volume-independent activation of colonic NBC by an increase in intracellular cAMP.