The small and large intestine secrete guanylin, a peptide homologous to heat stable enterotoxin (STa) elaborated by enterotoxigenic Escherichia coli. Guanylin's role in intestinal electrolyte transport was investigated in guanylin-deficient knockout mice and heterozygous littermate controls. Segments of mid-jejunum, distal ileum, and proximal and distal colon were studied in Ussing chambers in HCO3- Ringer under short circuit conditions. We found that (1) under basal conditions, all segments in control and knockout mice absorb Na+, and the knockout mouse proximal colon secretes Cl-; (2) all segments except the jejunum of knockout mice respond by increasing absorption in response to reductions in pH from 7.6 to 7.1; (3) all segments exhibit decreased absorption in response to 1 mM cAMP; (4) the jejunum and ileum of knockout and control mice, and the proximal colon of control mice (but not knockout mice) respond to the mucosal addition of 50 nM STa with decreases in absorption; and (5) mucosal guanylin caused similar decreases in proximal colon absorption in control and guanylin-deficient mice. These findings suggest that guanylin deficiency causes basal Cl- secretion and reduced responsiveness to STa in mouse proximal colon. The effectiveness of guanylin in this segment suggests a difference in the intestinal secretory actions of STa and guanylin.