1. Mucosally added synthetic guanylin and Escherichia coli heat-stable enterotoxin (STa) increased short-circuit current (ISC) across isolated muscle-stripped human intestine in vitro. 2. Serosal bumetanide inhibited ISC responses indicating that guanylin and STa stimulate electrogenic chloride secretion. 3. ISC responses were markedly greater in the colon than in the jejunum. 4. Pretreatment with indomethacin did not significantly alter the effects of guanylin and STa. 5. Both peptides induced concentration-dependent increases in the cyclic GMP content of human intestinal mucosa in vitro; cyclic AMP levels remained unchanged. 6. In contrast to ISC responses, increases in cyclic GMP content induced by guanylin and STa were markedly greater in the jejunum than in the colon. 7. Sodium nitroprusside (SNP) but not human alpha-atrial natriuratic peptide (CDD/ANP(99-126)) increased chloride secretion in human intestine; both agents induced small increases in intestinal cyclic GMP content. 8. Guanylin, STa and the nitric oxide (NO) donor SNP increased electrogenic chloride secretion across human intestinal mucosa in vitro by stimulation of cyclic GMP. The discrepancy between the effects on chloride secretion and intracellular cyclic GMP content suggest different cellular action sites of guanylin and STa in human small and large intestine.