Studies of full-thickness small intestinal preparations have shown that maximal anion secretion (indexed by short-circuit current, I_sc) during cAMP_i stimulation is transient and followed by a decline towards baseline. Declining I_sc is preceded by decreases in transepithelial conductance (G_t), which in the small intestine, reflects the lateral intercellular space (LIS) volume of the paracellular pathway. We hypothesized that decreases in LIS volume limit the magnitude and duration of cAMP_i-stimulated anion secretion. Experimental manipulations to increase the patency of the LIS (assessed by G_t and electron microscopy) were investigated for an effect on the magnitude of cAMP_i-stimulated anion secretion (assessed by the I_sc and isotopic fluxes) across murine small intestine. In control studies, changes of G_t after cAMP_i stimulation were associated with a morphological "collapse" of the LIS, which did not occur in intestine of cystic fibrosis transmembrane conductance regulator (CFTR)-null mice. Removal of the outer intestinal musculature, exposure to a serosal hypertonic solution or increased serosal hydrostatic pressure minimized reductions in G_t and increased the cAMP_i-stimulated I_sc response. Increased I_sc primarily resulted from increased Cl^- secretion that was largely bumetanide sensitive. However, bumetanide-insensitive I_sc was also increased and similar increases occurred in the Na⁺/K⁺/2Cl^- cotransporter NKCC1-null intestine, indicating that activities of non-NKCC1 anion uptake proteins are also affected by LIS volume. Thus, LIS patency is an important determinant of the magnitude and duration of CFTR-mediated anion secretion in murine small intestine. Decreases in LIS volume may limit the pool of available anions to basolateral transporters involved in transepithelial secretion.