C-type natriuretic peptide (CNP) is one of three members of the natriuretic peptide family. In the course of cloning frog CNP precursor cDNA, we found cDNA clones encoding a novel natriuretic peptide precursor. This novel natriuretic peptide precursor is a 118-residue peptide, carrying a 22-residue signal peptide at the N terminus and a 17-residue ring structure characteristic of the natriuretic peptide family at the C terminus. The precursor is highly homologous to mammalian CNP precursors. Its C-terminal Cys codon is directly followed by a termination codon. This feature is characteristic of CNP precursor cDNAs. The cGMP-stimulating activity of the novel natriuretic peptide was at the same level with those of frog CNP and mammalian CNPs, when rat CNP receptor was used in the cGMP assay system. Thus, this novel natriuretic peptide is structurally and functionally a member of the C-type natriuretic peptide. We designated the peptide as frog CNP II, and the previously described CNP as frog CNP I. By Northern blot analysis, expression of this novel CNP mRNA was found in brain, heart, lung, and stomach. On the other hand, frog CNP mRNA was mainly expressed in brain, where mammalian CNP mRNA is exclusively expressed. The distribution of the two frog CNPs suggests a complex system for regulating electrolyte and water balance in frog.