Guanylin and uroguanylin compose a family of natriuretic, diuretic, and kaliuretic peptides that bind to and activate apical membrane receptor guanylyl cyclase signaling molecules in renal and intestinal epithelia. Recently, a complementary DNA encoding an additional member of the guanylin family of cGMP-regulating peptides was isolated from lymphoid tissues of the opossum and was termed lymphoguanylin (LGN). A peptide analog of opossum LGN was synthesized containing a single disulfide bond with the internal cysteine-7 replaced by a serine residue (LGN^Cys7Ser7). The biological activity of LGN^Ser was tested by using a cGMP bioassay with cultured T84 (human intestinal) cells and opossum kidney (OK) cells. LGN^Ser has potencies and efficacies for activation of cGMP production in the intestinal and kidney cell lines that are 100- and 1,000-fold higher than LGN, respectively. In the isolated perfused rat kidney, LGN^Ser stimulated a maximal increase in fractional Na⁺ excretion from 24.8 ± 3.0 to 36.3 ± 3.3% 60 min after administration and enhanced urine flow from 0.15 ± 0.01 to 0.24 ± 0.01 ml · g¹ · min¹. LGN^Ser (0.69 µM) also increased fractional K⁺ excretion from 27.3 ± 2.3 to 38.0 ± 3.0% and fractional Cl excretion from 26.1 ± 0.8 to 43.5 ± 1.9. A ninefold increase in the urinary excretion of cGMP from 1.00 ± 0.04 to 9.28 ± 1.14 pmol/ml was elicited by LGN^Ser, whereas cAMP levels were not changed on peptide administration. These findings demonstrate that LGN^Ser, which contains a single disulfide bond like native LGN, activates guanylyl cyclase-C (GC-C) receptors in T84 and OK cells and may be very helpful in studying the physiological importance of activation of GC-C in vivo. LGN^Ser also exhibits full activity in the isolated perfused kidney equivalent to that observed previously with opossum uroguanylin, suggesting a physiological role for LGN in renal function. Thus the single amino acid substitution enhances the activity and potency of LGN.