Guanylin, a 15-amino acid peptide homologue of bacterial heat-stable enterotoxins, is an endogenous activator of guanylate cyclase C (GC-C). We isolated two novel guanylin molecules from rat intestinal mucosa. They contained guanylin-15 at their C-termini and were identified as guanylin-94 and guanylin-16 by amino acid sequencing and mass spectrometry. Guanylin-94 and guanylin-16 in total account for 85% of guanylin molecules in both the small and large intestine, guanylin-15 being a minor component. Rat guanylin-94 and guanylin-16 did not increase cyclic GMP production in T84 cells. Identification of the post-translational processing products of guanylin should provide a better understanding of the biosynthesis of the peptide.